Comparative transcriptional analysis of the satellite glial cell injury response

Satellite glial cells (SGCs) tightly surround and support primary sensory neurons in the peripheral nervous system and are increasingly recognized for their involvement in the development of neuropathic pain following nerve injury. The SGCs are difficult to investigate due to their flattened shape and tight physical connection to neurons in vivo and their rapid changes in phenotype and protein expression when cultured in vitro. Consequently, several aspects of SGC function under normal conditions as well as after a nerve injury remain to be explored. The recent advance in single cell RNAseq technologies has enabled a new approach to investigate SGCs. Here we publish a dataset from mice subjected to sciatic nerve injury as well as a dataset from dorsal root ganglia cells after 3 days in culture. We use a meta-analysis approach to compare the injury response with that in other published datasets and conclude that SGCs share a common signature following sciatic nerve crush and sciatic ligation, involving transcriptional regulation of cholesterol biosynthesis. We also observed a considerable transcriptional change when culturing SGCs, suggesting that some differentiate into a specialised in vitro state, while others start resembling Schwann cell-like precursors. The datasets are available via the Broad Institute Single Cell Portal.

Download Full-text

Presence and activation of pro-inflammatory macrophages are associated with CRYAB expression in vitro and after peripheral nerve injury

Journal of Neuroinflammation ◽

10.1186/s12974-021-02108-z ◽

2021 ◽

Vol 18 (1) ◽

Author(s):

Erin-Mai F. Lim ◽

Vahid Hoghooghi ◽

Kathleen M. Hagen ◽

Kunal Kapoor ◽

Ariana Frederick ◽

...

Keyword(s):

Sciatic Nerve ◽

Peripheral Nerve ◽

Nerve Injury ◽

Peripheral Nerve Injury ◽

Sciatic Nerve Crush ◽

Nerve Crush ◽

Cytokines And Chemokines ◽

Inflammatory Macrophages ◽

Nerve Crush Injury

Abstract Background Inflammation constitutes both positive and negative aspects to recovery following peripheral nerve injury. Following damage to the peripheral nervous system (PNS), immune cells such as macrophages play a beneficial role in creating a supportive environment for regrowing axons by phagocytosing myelin and axonal debris. However, a prolonged inflammatory response after peripheral nerve injury has been implicated in the pathogenesis of negative symptoms like neuropathic pain. Therefore, the post-injury inflammation must be carefully controlled to prevent secondary damage while allowing for regeneration. CRYAB (also known as alphaB-crystallin/HSPB5) is a small heat shock protein that has many protective functions including an immunomodulatory role in mouse models of multiple sclerosis, spinal cord injury, and stroke. Because its expression wanes and rebounds in the early and late periods respectively after PNS damage, and CRYAB null mice with sciatic nerve crush injury display symptoms of pain, we investigated whether CRYAB is involved in the immune response following PNS injury. Methods Sciatic nerve crush injuries were performed in age-matched Cryab knockout (Cryab−/−) and wildtype (WT) female mice. Nerve segments distal to the injury site were processed by immunohistochemistry for macrophages and myelin while protein lysates of the nerves were analyzed for cytokines and chemokines using Luminex and enzyme-linked immunosorbent assay (ELISA). Peritoneal macrophages from the two genotypes were also cultured and polarized into pro-inflammatory or anti-inflammatory phenotypes where their supernatants were analyzed for cytokines and chemokines by ELISA and protein lysates for macrophage antigen presenting markers using western blotting. Results We report that (1) more pro-inflammatory CD16/32+ macrophages are present in the nerves of Cryab−/− mice at days 14 and 21 after sciatic nerve crush-injury compared to WT counterparts, and (2) CRYAB has an immunosuppressive effect on cytokine secretion [interleukin (IL)-β, IL-6, IL-12p40, tumor necrosis factor (TNF)-α] from pro-inflammatory macrophages in vitro. Conclusions CRYAB may play a role in curbing the potentially detrimental pro-inflammatory macrophage response during the late stages of peripheral nerve regeneration.

Download Full-text

204 Voluntary Exercise Modulates Macrophage Polarization Following Sciatic Nerve Injury and Improves Functional Recovery in Mice

Neurosurgery ◽

10.1093/neuros/nyx417.204 ◽

2017 ◽

Vol 64 (CN_suppl_1) ◽

pp. 255-255

Author(s):

Megan M Jack ◽

Douglas E Wright

Keyword(s):

Sciatic Nerve ◽

Peripheral Nerve ◽

Nerve Injury ◽

Functional Recovery ◽

Macrophage Polarization ◽

Sciatic Nerve Crush ◽

Nerve Crush ◽

Sciatic Nerves ◽

Running Wheels

Abstract INTRODUCTION Peripheral nerve injury is associated with trauma and is often amenable to surgery. Functional recovery remains a challenging clinical problem that often leads to significant morbidity. Therapies that augment surgical repair may be beneficial in functional outcomes. Macrophages are responsible for the breakdown of debris following injury as well as promotion of regenerative signals. Macrophage polarization is the process by which macrophages take on phenotypically distinct functions based on the local environment and signaling cues. Exercise has been shown to drive macrophage polarization from a pro-inflammatory M1 phenotype towards an anti-inflammatory M2 phenotype in numerous tissues, but remains uninvestigated in the peripheral nervous system. METHODS The purpose of our study was to identify how exercise affects macrophage polarization, motor and sensory function, and neuroregeneration following sciatic nerve crush. Male and female C57BL/6 mice underwent sciatic nerve crush injury and were then given access to running wheels (exercised) or not given access to running wheels (sedentary) for 4 weeks. Analysis included behavioral assessments, anatomical studies, and in vitro studies. RESULTS >Exercised mice ran an average of 2.9 km per night. Injured exercised mice were protected from the development of thermal hyperalgesia. Exercised mice had fewer paw slips on beam walk testing compared to sedentary mice. No differences were measured in mechanical sensitivity or motor coordination and balance. Motor nerve conduction velocities from injured exercised animals were significantly higher than injured sedentary animals suggesting improved nerve recovery with exercise. Injured sciatic nerves from exercised mice demonstrated increased M2 macrophages compared to sciatic nerves from injured sedentary mice. The behavioral changes and altered macrophage polarization correlated with increased epidermal nerve fiber density, improved myelination, and increased in vitro neurite outgrowth from injured exercised animals. CONCLUSION Exercise alters macrophage polarization towards an anti-inflammatory phenotype which improves repair and recovery of the injured peripheral nerve.

Download Full-text

Circ-Spidr enhances axon regeneration after peripheral nerve injury

Cell Death and Disease ◽

10.1038/s41419-019-2027-x ◽

2019 ◽

Vol 10 (11) ◽

Cited By ~ 3

Author(s):

Susu Mao ◽

Tao Huang ◽

Yuanyuan Chen ◽

Longxiang Shen ◽

Shuoshuo Zhou ◽

...

Keyword(s):

Nervous System ◽

Sciatic Nerve ◽

Nerve Injury ◽

Axon Regeneration ◽

Neuronal Injury ◽

Circular Rnas ◽

Sciatic Nerve Crush ◽

Drg Neurons ◽

Nerve Crush

Abstract Accumulating evidence suggests that circular RNAs (circRNAs) are abundant and play critical roles in the nervous system. However, their functions in axon regeneration after neuronal injury are unclear. Due to its robust regeneration capacity, peripheral nervous system is ideal for seeking the regulatory circRNAs in axon regeneration. In the present work, we obtained an expression profile of circRNAs in dorsal root ganglions (DRGs) after rat sciatic nerve crush injury by RNA sequencing (RNA-Seq) and found the expression level of circ-Spidr was obviously increased using quantitative real-time polymerase chain reaction (qRT-PCR). Furthermore, circ-Spidr was proved to be a circular RNA enriched in the cytoplasm of DRG neurons. Through in vitro and in vivo experiments, we determined that down-regulation of circ-Spidr could suppress axon regeneration of DRG neurons after sciatic nerve injury partially through modulating PI3K-Akt signaling pathway. Together, our results reveal a crucial role for circRNAs in regulating axon regeneration after neuronal injury which may further serve as a potential therapeutic avenue for neuronal injury repair.

Download Full-text

3D-printed nerve guidance conduits multi-functionalized with canine multipotent mesenchymal stromal cells promote neuroregeneration after sciatic nerve injury in rats

Stem Cell Research & Therapy ◽

10.1186/s13287-021-02315-8 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Diego Noé Rodríguez-Sánchez ◽

Giovana Boff Araujo Pinto ◽

Luciana Politti Cartarozzi ◽

Alexandre Leite Rodrigues de Oliveira ◽

Ana Livia Carvalho Bovolato ◽

...

Keyword(s):

Growth Factor ◽

Sciatic Nerve ◽

Nerve Injury ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Multipotent Mesenchymal Stromal Cells ◽

Sciatic Nerve Injury ◽

Motor Deficits ◽

3D Printed

Abstract Background Nerve injuries are debilitating, leading to long-term motor deficits. Remyelination and axonal growth are supported and enhanced by growth factor and cytokines. Combination of nerve guidance conduits (NGCs) with adipose-tissue-derived multipotent mesenchymal stromal cells (AdMSCs) has been performing promising strategy for nerve regeneration. Methods 3D-printed polycaprolactone (PCL)-NGCs were fabricated. Wistar rats subjected to critical sciatic nerve damage (12-mm gap) were divided into sham, autograft, PCL (empty NGC), and PCL + MSCs (NGC multi-functionalized with 106 canine AdMSCs embedded in heterologous fibrin biopolymer) groups. In vitro, the cells were characterized and directly stimulated with interferon-gamma to evaluate their neuroregeneration potential. In vivo, the sciatic and tibial functional indices were evaluated for 12 weeks. Gait analysis and nerve conduction velocity were analyzed after 8 and 12 weeks. Morphometric analysis was performed after 8 and 12 weeks following lesion development. Real-time PCR was performed to evaluate the neurotrophic factors BDNF, GDNF, and HGF, and the cytokine and IL-10. Immunohistochemical analysis for the p75NTR neurotrophic receptor, S100, and neurofilament was performed with the sciatic nerve. Results The inflammatory environment in vitro have increased the expression of neurotrophins BDNF, GDNF, HGF, and IL-10 in canine AdMSCs. Nerve guidance conduits multi-functionalized with canine AdMSCs embedded in HFB improved functional motor and electrophysiological recovery compared with PCL group after 12 weeks. However, the results were not significantly different than those obtained using autografts. These findings were associated with a shift in the regeneration process towards the formation of myelinated fibers. Increased immunostaining of BDNF, GDNF, and growth factor receptor p75NTR was associated with the upregulation of BDNF, GDNF, and HGF in the spinal cord of the PCL + MSCs group. A trend demonstrating higher reactivity of Schwann cells and axonal branching in the sciatic nerve was observed, and canine AdMSCs were engrafted at 30 days following repair. Conclusions 3D-printed NGCs multi-functionalized with canine AdMSCs embedded in heterologous fibrin biopolymer as cell scaffold exerted neuroregenerative effects. Our multimodal approach supports the trophic microenvironment, resulting in a pro-regenerative state after critical sciatic nerve injury in rats.

Download Full-text

Continuation of fast axonal transport in regenerating axons in vitro

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y79-189 ◽

1979 ◽

Vol 57 (11) ◽

pp. 1251-1255

Author(s):

M. A. Bisby ◽

C. E. Hilton

Keyword(s):

Sciatic Nerve ◽

Vagus Nerve ◽

Axonal Transport ◽

Nerve Injury ◽

Axon Regeneration ◽

Free Medium ◽

Fast Axonal Transport ◽

Sensory Axons

A previous study by McLean and co-workers reported that regenerating axons of the rabbit vagus nerve were unable to sustain axonal transport in vitro for several months after nerve injury. In contrast, we found that sensory axons of the rat sciatic nerve were able to transport 3H-labeled protein into their regenerating portions distal to the site of injury within a week after injury when placed in vitro. Transport in vitro was not significantly less than transport in axons maintained in vivo for the same period. Transport occurred in the medium that was used by the McLean group, but was significantly reduced in calcium-free medium. When axon regeneration was delared, only small amounts of activity were present in the nerve distal to the site of injury, showing that labeled protein normally present in that part of the nerve was associated with axons and was not a result of local precursor uptake by nonneural elements in the sciatic nerve. We were not able to explain the failure of McLean and co-workers to demonstrate transport in vitro in regenerating vagus nerve, but we conclude that there is no general peculiarity of growing axons that makes them unable to sustain transport in vitro.

Download Full-text

Preparation of Encapsulated Resveratrol Liposome Thermosensitive Gel and Evaluation of Its Capability to Repair Sciatic Nerve Injury in Rats

Journal of Nanomaterials ◽

10.1155/2020/2840162 ◽

2020 ◽

Vol 2020 ◽

pp. 1-13

Author(s):

Hui Yao ◽

Hao Lu ◽

Rong Zou ◽

Xiwen Chen ◽

Hanlin Xu

Keyword(s):

Sciatic Nerve ◽

Nerve Injury ◽

Drug Loading ◽

Particle Diameter ◽

Sciatic Nerve Injury ◽

Release Time ◽

Temperature Sensitive ◽

Polymer Gel ◽

Injury Model

The purpose of this study was to prepare a liposomal temperature-sensitive gel able to slowly release resveratrol after local intramuscular injection. The best formulation of resveratrol liposomes was based on the highest encapsulation efficiency and drug loading designed by Box-Behnken. The prepared liposomes were approximately circular, with a mean particle diameter of 161.5±0.12 nm and zeta potential of -6.9 mV. The optimized liposomes were dispersed in a polymer gel (PLGA-PEG-PLGA) for preparation of an in situ-formed gel at 35±2°C. In vitro release of the prepared liposome temperature-sensitive gel was studied and compared with ordinary drug-releasing gels, revealing a significantly longer drug release time. Finally, a rat sciatic nerve injury model was used to evaluate the pharmacological activity of the liposome temperature-sensitive gels for the repair of damaged nerves. The results indicate that the gel was able to promote recovery of damaged nerves.

Download Full-text

Reduced Renshaw Recurrent Inhibition after Neonatal Sciatic Nerve Crush in Rats

Neural Plasticity ◽

10.1155/2014/786985 ◽

2014 ◽

Vol 2014 ◽

pp. 1-11

Author(s):

Liang Shu ◽

Jingjing Su ◽

Lingyan Jing ◽

Ying Huang ◽

Yu Di ◽

...

Keyword(s):

Spinal Cord ◽

Sciatic Nerve ◽

Peripheral Nerve ◽

Nerve Injury ◽

Peripheral Nerve Injury ◽

Recurrent Inhibition ◽

Crush Injury ◽

Sciatic Nerve Crush ◽

Nerve Crush ◽

Nerve Crush Injury

Renshaw recurrent inhibition (RI) plays an important gated role in spinal motion circuit. Peripheral nerve injury is a common disease in clinic. Our current research was designed to investigate the change of the recurrent inhibitory function in the spinal cord after the peripheral nerve crush injury in neonatal rat. Sciatic nerve crush was performed on 5-day-old rat puppies and the recurrent inhibition between lateral gastrocnemius-soleus (LG-S) and medial gastrocnemius (MG) motor pools was assessed by conditioning monosynaptic reflexes (MSR) elicited from the sectioned dorsal roots and recorded either from the LG-S and MG nerves by antidromic stimulation of the synergist muscle nerve. Our results demonstrated that the MSR recorded from both LG-S or MG nerves had larger amplitude and longer latency after neonatal sciatic nerve crush. The RI in both LG-S and MG motoneuron pools was significantly reduced to virtual loss (15–20% of the normal RI size) even after a long recovery period upto 30 weeks after nerve crush. Further, the degree of the RI reduction after tibial nerve crush was much less than that after sciatic nerve crush indicatig that the neuron-muscle disconnection time is vital to the recovery of the spinal neuronal circuit function during reinnervation. In addition, sciatic nerve crush injury did not cause any spinal motor neuron loss but severally damaged peripheral muscle structure and function. In conclusion, our results suggest that peripheral nerve injury during neonatal early development period would cause a more sever spinal cord inhibitory circuit damage, particularly to the Renshaw recurrent inhibition pathway, which might be the target of neuroregeneration therapy.

Download Full-text

Lack of correlation between spinal microgliosis and long-term development of tactile hypersensitivity in two different sciatic nerve crush injury

Molecular Pain ◽

10.1177/17448069211011326 ◽

2021 ◽

Vol 17 ◽

pp. 174480692110113

Author(s):

Hyoung Woo Kim ◽

Chan Hee Won ◽

Seog Bae Oh

Keyword(s):

Spinal Cord ◽

Chronic Pain ◽

Sciatic Nerve ◽

Peripheral Nerve ◽

Nerve Injury ◽

Peripheral Nerve Injury ◽

Sensory Function ◽

Crush Injury ◽

Sciatic Nerve Crush

Microglia activation following peripheral nerve injury has been shown to contribute to central sensitization of the spinal cord for the development of neuropathic pain. In a recent study, we reported that the amount of nerve damage does not necessarily correlate with chronic pain development. Here we compared the response of spinal microglia, using immunohistochemistry as a surrogate of microglial activation, in mice with two different types of crush injury of the sciatic nerve. We confirmed that incomplete crush of the sciatic nerve (partial crush injury, PCI) resulted in tactile hypersensitivity after the recovery of sensory function (15 days after surgery), whereas the hypersensitivity was not observed after the complete crush (full crush injury, FCI). We observed that immunoreactivity for Iba-1, a microglial marker, was greater in the ipsilateral dorsal horn of lumbar (L4) spinal cord of mice 2 days after FCI compared to PCI, positively correlating with the intensity of crush injury. Ipsilateral Iba-1 reactivity was comparable between injuries at 7 days with a significant increase compared to the contralateral side. By day 15 after injury, ipsilateral Iba-1 immunoreactivity was much reduced compared to day 7 and was not different between the groups. Our results suggest that the magnitude of the early microgliosis is dependent on injury severity, but does not necessarily correlate with the long-term development of chronic pain-like hypersensitivity after peripheral nerve injury.

Download Full-text

BDNF rescues myosin heavy chain IIB muscle fibers after neonatal nerve injury

AJP Cell Physiology ◽

10.1152/ajpcell.00583.2003 ◽

2004 ◽

Vol 287 (1) ◽

pp. C22-C29 ◽

Cited By ~ 34

Author(s):

Kambiz Mousavi ◽

David J. Parry ◽

Bernard J. Jasmin

Keyword(s):

Sciatic Nerve ◽

Myosin Heavy Chain ◽

Nerve Injury ◽

Motor Neurons ◽

Heavy Chain ◽

Neurotrophic Factor ◽

Muscle Fibers ◽

Sciatic Nerve Crush ◽

Nerve Crush ◽

Combined Administration

Neonatal sciatic nerve injury is known to result in an extensive loss of lumbar motor neurons as well as the disappearance of their respective muscle fibers in the hindlimb musculature. The loss of motor neurons and muscle fibers can be prevented by immediate administration of target-derived neurotrophic factors to the site of injury. In the present study, we investigated the role of ciliary neurotrophic factor (CNTF) and brain-derived neurotrophic factor (BDNF) in the survival and maturation of a subset of motor neurons innervating the extensor digitorum longus (EDL) and tibialis anterior (TA) muscles. We have shown that combined administration of CNTF and BDNF prevented the loss of motor units after neonatal nerve injury and contributed to the maintenance of muscle mass. Importantly, this combined neurotrophin regimen also prevented the disappearance of muscle fibers that express myosin heavy chain IIB (MyHC IIB) in both EDL and TA muscles 3 mo after neonatal sciatic nerve crush. In parallel studies, we observed a higher level of BDNF in EDL muscle during the critical period of development when motor neurons are highly susceptible to target removal. Given our previous findings that combined administration of CNTF with neurotrophin-3 (NT-3) or neurotrophin-4/5 (NT-4/5) did not result in the rescue of MyHC IIB fibers in EDL, the present results show the importance of muscle-derived BDNF in the survival and maturation of a subpopulation of motor neurons and of MyHC IIB muscle fibers during neonatal development of the neuromuscular system.

Download Full-text

Calcium mediated functional interplay between myocardial cells upon laser-induced single-cell injury: an in vitro study of cardiac cell death signaling mechanisms

Cell Communication and Signaling ◽

10.1186/s12964-020-00689-5 ◽

2020 ◽

Vol 18 (1) ◽

Author(s):

Krishna Chander Sridhar ◽

Nils Hersch ◽

Georg Dreissen ◽

Rudolf Merkel ◽

Bernd Hoffmann

Keyword(s):

Cell Death ◽

Single Cell ◽

Cell Injury ◽

Specific Effect ◽

Myocardial Cell ◽

Myocardial Tissue ◽

Cell Clusters ◽

Signaling Events ◽

Intracellular Ca

Abstract Background The electromechanical function of myocardial tissue depends on the intercellular communication between cardiomyocytes (CMs) as well as their crosstalk with other cell types. Cell injury, and subsequent death trigger inflammation as in myocardial infarction (MI) resulting in myocardial remodeling. Although mechanisms underlying myocardial cell death have been studied so far, the signaling events following single cell death and spontaneous response of connected cells in the myocardial tissue is still barely understood. Methods Here, we investigated the effect of laser-induced single cell death on Calcium (Ca2+) concentrations and transport in myocardial cell clusters in vitro. Spatial and temporal changes in intracellular Ca2+ concentrations [Ca2+]i were studied using a fluorescent calcium indicator, Fluo-4AM. Spontaneous signaling events following cell death were studied in rat embryonic cardiomyocytes and non-myocytes using separate cell culture systems. Results Cell death triggered spontaneous increase in intracellular Ca2+ levels ([Ca2+]i) of surrounding cells. The spread of the observed propagating Ca2+ signal was slow and sustained in myocytes while it was rapid and transient in fibroblasts (Fbs). Further, sustained high Ca2+ levels temporarily impaired the contractility in CMs. The cell-type specific effect of ablation was confirmed using separate cultures of CMs and Fbs. Comparing Ca2+ propagation speed in myocytes and fibroblasts, we argue for a diffusion-driven Ca2+ propagation in myocytes, but not in fibroblasts. Radial and sequential Ca2+ diffusion across the CMs through cell–cell contacts and presence of Cx43-based intercellular junctions indicated a gap junction flow of Ca2+. Conclusions These findings illustrate the spontaneous Ca2+-mediated functional interplay in myocardial cell clusters upon mechanical injury and, further, the difference in Ca2+ signaling in cardiomyocytes and fibroblasts.

Download Full-text