Immunohistochemical, Ultrastructural and Functional Analysis of Axonal Regeneration through Peripheral Nerve Grafts Containing Schwann Cells Expressing BDNF, CNTF or NT3

Abstract

We used morphological, immunohistochemical and functional assessments to determine the impact of genetically-modified peripheral nerve (PN) grafts on axonal regeneration after injury. Grafts were assembled from acellular nerve sheaths repopulated ex vivo with Schwann cells (SCs) modified to express brain-derived neurotrophic factor (BDNF), a secretable form of ciliary neurotrophic factor (CNTF), or neurotrophin-3 (NT3). Grafts were used to repair unilateral 1 cm defects in rat peroneal nerves and 10 weeks later outcomes were compared to normal nerves and various controls: autografts, acellular grafts and grafts with unmodified SCs. The number of regenerated βIII-Tubulin positive axons was similar in all grafts with the exception of CNTF, which contained the fewest immunostained axons. There were significantly lower fiber counts in acellular, untransduced SC and NT3 groups using a PanNF antibody, suggesting a paucity of large caliber axons. In addition, NT3 grafts contained the greatest number of sensory fibres, identified with either IB4 or CGRP markers. Examination of semi- and ultra-thin sections revealed heterogeneous graft morphologies, particularly in BDNF and NT3 grafts in which the fascicular organization was pronounced. Unmyelinated axons were loosely organized in numerous Remak bundles in NT3 grafts, while the BDNF graft group displayed the lowest ratio of umyelinated to myelinated axons. Gait analysis revealed that stance width was increased in rats with CNTF and NT3 grafts, and step length involving the injured left hindlimb was significantly greater in NT3 grafted rats, suggesting enhanced sensory sensitivity in these animals. In summary, the selective expression of BDNF, CNTF or NT3 by genetically modified SCs had differential effects on PN graft morphology, the number and type of regenerating axons, myelination, and locomotor function.

c-Jun Gene-Modified Schwann Cells: Upregulating Multiple Neurotrophic Factors and Promoting Neurite Outgrowth

“…However, an increasing number of studies have shown that co-delivery of multiple factors, rather than a single factor, holds greater therapeutic promise for promoting nerve regeneration because peripheral nerves contained different neuronal and glial subpopulations. 4,20,22,23 In the present study, overexpression of the c-Jun gene in SCs significantly increased the expression and secretion of multiple NFs, including GDNF, BDNF, Artn, LIF, and NGF. In addition, the c-Junmodified SCs significantly increased the average length of axons and the average area occupied by DRG axons in the coculture system, highlighting their potential to promote neuronal survival, axonal regeneration, and functional recovery in response to PNS injury.…”

“…17,18,34 Similarly, overexpression of NGF and BDNF in SCs also has beneficial effects for peripheral nerve repair. 19,20 Artn exerts multiple effects on sensory neuron generation, survival, regeneration, and functional recovery. [38][39][40][41] LIF has also been shown to play a positive role in nerve regeneration.…”

“…10,11 Schwann cells (SCs), which play a pivotal role during nerve regeneration in vivo, are an attractive type of transplant cell with potentiated functionality due to the expression of multiple neurotrophic factors (NFs), the organization of Büngner bands, and the ability to form myelin. [12][13][14] Recent studies have revealed that delivery of SCs to NCs is advantageous for nerve repair, particularly when using SCs that overexpress an NF, such as basic fibroblast growth factor (FGF-2), 15,16 glial cell line-derived neurotrophic factor (GDNF), 17,18 nerve growth factor (NGF), 19 brainderived neurotrophic factor (BDNF), 20 or neurotrophin-3 (NT-3). 21 However, an increasing number of studies have indicated that the application of multiple NFs, rather than a single type, holds greater therapeutic promise.…”

Towards the development of a bioengineered uterus: Comparison of different protocols for rat uterus decellularization

“…Various techniques for decellularization and recellularization have been examined for pre-clinical interventions of miscellaneous tissues, e.g., small intestine [13,14], urinary bladder [15], heart valves [16], larynx [17], dermis [18], musculofascial tissue [19] and nerves [20,21]. Concerning the female internal genital tract, small patches of myometrial tissue were recently decellularized using a modified protocol that had been optimized for decellularization of blood vessels [22].…”

Development of a cell-based treatment for long-term neurotrophin expression and spiral ganglion neuron survival

“…Schwann cells and fibroblasts were isolated from rat sciatic nerve explants using established protocols (Morrissey et al, 1991;Plant et al, 2002;Godinho et al, 2013). Briefly, 8-10-week-old rats were killed using sodium pentobarbitone (150 mg/kg intraperitoneally) and the sciatic nerves removed.…”