The Response of Dorsal Root Ganglion Axons to Nerve Growth Factor Gradients Depends on Spinal Level

Laser refractive surgeries reshape corneal stroma to correct refractive errors, but unavoidably affect corneal nerves. Slow nerve regeneration and atypical neurite morphology cause desensitization and neuro-epitheliopathy. Following injury, surviving corneal stromal keratocytes (CSKs) are activated to stromal fibroblasts (SFs). How these two different cell types influence nerve regeneration is elusive. Our study evaluated the neuro-regulatory effects of human SFs versus CSKs derived from the same corneal stroma using an in vitro chick dorsal root ganglion model. The neurite growth was assessed by a validated concentric circle intersection count method. Serum-free conditioned media (CM) from SFs promoted neurite growth dose-dependently, compared to that from CSKs. We detected neurotrophic and pro-inflammatory factors (interleukin-8, interleukin-15, monocyte chemoattractant protein-1, eotaxin, RANTES) in SFCM by Bio-Plex Human Cytokine assay. More than 130 proteins in SFCM and 49 in CSKCM were identified by nanoLC-MS/MS. Proteins uniquely present in SFCM had reported neuro-regulatory activities and were predicted to regulate neurogenesis, focal adhesion and wound healing. Conclusively, this was the first study showing a physiological relationship between nerve growth and the metabolically active SFs versus quiescent CSKs from the same cornea source. The dose-dependent effect on neurite growth indicated that nerve regeneration could be influenced by SF density.

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“…NGF was selected as the reference neurotrophin due to its high bioactivity on DRG explant cultures37. As depicted in Fig.…”

Section: Resultsmentioning

confidence: 99%

Nerve regeneration by human corneal stromal keratocytes and stromal fibroblasts

Yam

Williams

Setiawan

et al. 2017

Sci Rep

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“…This process helps to construct a dynamic NTF gradient that guides axons to the distal target. Thus, NTFs not only provide growth-promoting (trophic) effects to the neurite but also exert chemotactic (tropic) effects on the same neurite (35,53,54). In the present study, nerve regeneration was evaluated at 2 and 3 wk after cell transplantation.…”

Section: Discussionmentioning

confidence: 98%

Time‐restricted release of multiple neurotrophic factors promotes axonal regeneration and functional recovery after peripheral nerve injury

et al. 2019

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Delivery of multiple neurotrophic factors (NTFs), especially with time‐restricted release kinetics, holds great potential for nerve repair. In this study, we utilized the tetracycline‐regulatable Tet‐On 3G system to control the expression of c‐Jun, which is a common regulator of multiple NTFs in Schwann cells (SCs). In vitro, Tet‐On/c‐Jun‐modified SCs showed a tightly controllable secretion of multiple NTFs, including glial cell line‐derived NTF, nerve growth factor, brain‐derived NTF, and artemin, by the addition or removal of doxycycline (Dox). When Tet‐On/c‐Jun‐transduced SCs were grafted in vivo, the expression of NTFs could also be regulated by oral administration or removal of Dox. Fluoro‐Gold retrograde tracing results indicated that a biphasic NTF expression scheme (Dox+3/−9, NTFs were up‐regulated for 3 wk and declined to physiologic levels for another 9 wk) achieved more axonal regeneration than continuous up‐regulation of NTFs (Dox+12) or no NTF induction (Dox−12). More importantly, the Dox+3/−9–group animals showed much better functional recovery than the animals in the Dox+12 and Dox−12 groups. Our findings, for the first time, demonstrated drug‐controllable expression of multiple NTFs in nerve repair cells both in vitro and in vivo. These findings provide new hope for developing an optimal therapeutic alternative for nerve repair through the time‐restricted release of multiple NTFs using Tet‐On/c‐Jun‐modified SCs.—Huang, L., Xia, B., Shi, X., Gao, J., Yang, Y., Xu, F., Qi, F., Liang, C., Huang, J., Luo, Z. Time‐restricted release of multiple neurotrophic factors promotes axonal regeneration and functional recovery after peripheral nerve injury. FASEB J. 33, 8600–8613 (2019). http://www.fasebj.org

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“…The Wake Forest Institutional Animal Care and Use Committee (WF IACUC) provided us a waiver from ethics board review. Since the response of DRGs to identical concentrations or gradients of neurotrophic factors is known to significantly vary depending on their position along the rostro-caudal axis of the body [ 4 ], we limited our assays to the ganglia from the lumbar spinal levels. Upon isolation, they were briefly washed in sterile PBS and then immediately collected in the base medium (DMEM/HG with 2% horse serum and 1% penicillin/streptomycin) until plating in collagen gel for the guidance assay.…”

Section: Methodsmentioning

confidence: 99%

“…These factors are expressed endogenously by Schwann glial cells and other cell types in response to the neural injury, but this expression is often insufficient to sustain full regeneration and reinnervation [2]. In regenerative medicine, it is often desirable to not only enhance the neural regeneration in general, but to specifically direct the growing neurites towards a desired target [3,4].…”

Section: Introductionmentioning

confidence: 99%

“…The first step in identifying the potential signaling molecules with the capacity to direct neurite growth is to screen them in chemotaxis assays. In spite of the progress in the field, many of the available guidance assays have been showing limitations regarding controlling the precise concentration of signaling molecules in the gradient field and/or are difficult to implement and reproduce as they involve highly specialized equipment or custom devices manufactured in individual labs [3][4][5]. In this study, we developed a simple guidance assay that allows to assess the effect of neurotrophic factors on neurite outgrowth from dorsal root ganglia (DRGs) using only standard tissue culture materials.…”

Section: Introductionmentioning

confidence: 99%

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Synergistic effect of CNTF and GDNF on directed neurite growth in chick embryo dorsal root ganglia

et al. 2020

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It is often critical to improve the limited regenerative capacity of the peripheral nerves and direct neural growth towards specific targets, such as surgically implanted bioengineered constructs. One approach to accomplish this goal is to use extrinsic neurotrophic factors. The candidate factors first need to be identified and characterized in in vitro tests for their ability to direct the neurite growth. Here, we present a simple guidance assay that allows to assess the chemotactic effect of signaling molecules on the growth of neuronal processes from dorsal root ganglia (DRG) using only standard tissue culture materials. We used this technique to quantitatively determine the combined and individual effects of the ciliary neurotrophic factor (CNTF) and glial cell line-derived neurotrophic factor (GDNF) on neurite outgrowth. We demonstrated that these two neurotrophic factors, when applied in a 1:1 combination, but not individually, induced directed growth of neuronal processes towards the source of the gradient. This chemotactic effect persists without significant changes over a wide (10-fold) concentration range. Moreover, we demonstrated that other, more general growth parameters that do not evaluate growth in a specific direction (such as, neurite length and trajectory) were differentially affected by the concentration of the CNTF/GNDF mixture. Furthermore, GDNF, when applied individually, did not have any chemotactic effect, but caused significant neurite elongation and an increase in the number of neurites per ganglion.

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The Response of Dorsal Root Ganglion Axons to Nerve Growth Factor Gradients Depends on Spinal Level

Cited by 15 publications

References 30 publications

Nerve regeneration by human corneal stromal keratocytes and stromal fibroblasts

Nerve regeneration by human corneal stromal keratocytes and stromal fibroblasts

Time‐restricted release of multiple neurotrophic factors promotes axonal regeneration and functional recovery after peripheral nerve injury

Synergistic effect of CNTF and GDNF on directed neurite growth in chick embryo dorsal root ganglia

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