Immortalization of human normal and NF1 neurofibroma Schwann cells.

Neurofibromas, which are benign Schwann cell tumors, are the hallmark feature in the autosomal dominant condition neurofibromatosis 1 (NF1) and are associated with biallelic loss of NF1 gene function. There is a need for effective therapies for neurofibromas, particularly the larger, plexiform neurofibromas. Tissue culture is an important tool for research. However, it is difficult to derive enriched human Schwann cell cultures, and most enter replicative senescence after 6-10 passages, impeding cell-based research in NF1. Through exogenous expression of human telomerase reverse transcriptase and murine cyclin-dependent kinase (mCdk4), normal (NF1 wild-type), neurofibroma-derived Schwann cells heterozygous for NF1 mutation, and neurofibroma-derived Schwann cells homozygous for NF1 mutation were immortalized, including some matched samples from the same NF1 patient. Initial experiments employed retroviral vectors, while subsequent work utilized lentiviral vectors carrying these genes because of improved efficiency. Expression of both transgenes was required for immortalization. Molecular and immunohistochemical analysis indicated that these cell lines are of Schwann cell lineage and have a range of phenotypes, many of which are consistent with their primary cultures. This is the first report of immortalization and detailed characterization of multiple human NF1 normal nerve and neurofibroma-derived Schwann cell lines, which will be highly useful research tools to study NF1 and other Schwann tumor biology and conditions.

Metalloproteinase-dependent predegeneration in vitro enhances axonal regeneration within acellular peripheral nerve grafts.

Injury to peripheral nerve initiates a degenerative process that converts the denervated nerve from a suppressive environment to one that promotes axonal regeneration. We investigated the role of matrix metalloproteinases (MMPs) in this degenerative process and whether effective predegenerated nerve grafts could be produced in vitro. Rat peripheral nerve explants were cultured for 1-7 d in various media, and their neurite-promoting activity was assessed by cryoculture assay, in which neurons are grown directly on nerve sections. The neurite-promoting activity of cultured nerves increased rapidly and, compared with uncultured nerve, a maximum increase of 72% resulted by 2 d of culture in the presence of serum. Remarkably, the neurite-promoting activity of short-term cultured nerves was also significantly better than nerves degenerated in vivo. We examined whether in vitro degeneration is MMP dependent and found that the MMP inhibitor N-[(2R)-2(hydroxamidocarbonylmethyl)-4-methylpantanoyl]-l-tryptophan methylamide primarily blocked the degenerative increase in neurite-promoting activity. In the absence of hematogenic macrophages, MMP-9 was trivial, whereas elevated MMP-2 expression and activation paralleled the increase in neurite-promoting activity. MMP-2 immunoreactivity localized to Schwann cells and the endoneurium and colocalized with gelatinolytic activity as demonstrated by in situ zymography. Finally, in vitro predegenerated nerves were tested as acellular grafts and, compared with normal acellular nerve grafts, axonal ingress in vivo was approximately doubled. We conclude that Schwann cell expression of MMP-2 plays a principal role in the degenerative process that enhances the regeneration-promoting properties of denervated nerve. Combined with their low immunogenicity, acellular nerve grafts activated by in vitro predegeneration may be a significant advancement for clinical nerve allografting.

Nerve grafts with various sensory and motor fiber compositions are equally effective for the repair of a mixed nerve defect.

Autologous, cellular nerve grafts are commonly used to bridge nerve gaps in the clinical setting. Sensory nerves are most often selected for autografting because of their relative ease of procurement and low donor site morbidity. A series of recent reports conclude that sensory isografts are inferior to motor and mixed nerve isografts for the repair of a mixed nerve defect in rat. The aim of the present study was to determine if the disparity reported with cellular graft subtypes exists for detergent decellularized, chondroitinase ABC processed nerve grafts. We hypothesized that processing removes or neutralizes the inferior properties attributed to sensory nerve grafts. Saphenous (cutaneous branch), femoral quadriceps (muscle branch) and tibial (mixed trunk) nerve grafts 5 mm in length were used in tensionless reconstruction of syngenic rat tibial nerves. Nerve regeneration through the grafts and into the recipient distal nerve was evaluated 21 days after grafting by two methods, toluidine blue staining of semi-thin sections (myelinated axons) and neurofilament-immunolabeling (total axons). Contrary to previous reports using this grafting scheme, we found no significant difference in the myelinated axon counts for the three cellular graft subtypes. Moreover, total axon counts indicated cellular saphenous nerve grafts were more effective than the quadriceps and tibial nerve grafts. A similar though less pronounced trend was found for the decellularized processed grafts. These findings indicate that nerve graft composition (sensory and motor) has no substantial impact on the short-term outcome of nerve regeneration in a mixed nerve repair model.

Chondroitinase treatment increases the effective length of acellular nerve grafts.

Acellular nerve allografts have been explored as an alternative to nerve autografting. It has long been recognized that there is a distinct limit to the effective length of conventional acellular nerve grafts, which must be overcome for many grafting applications. In rodent models nerve regeneration fails in acellular nerve grafts greater than 2 cm in length. In previous studies we found that nerve regeneration is markedly enhanced with acellular nerve grafts in which growth-inhibiting chondroitin sulfate proteoglycan was degraded by pretreatment with chondroitinase ABC (ChABC). Here, we tested if nerve regeneration can be achieved through 4-cm acellular nerve grafts pretreated with ChABC. Adult rats received bilateral sciatic nerve segmental resection and repair with a 4 cm, thermally acellularized, nerve graft treated with ChABC (ChABC graft) or vehicle-treated acellularized graft (Control graft). Nerve regeneration was examined 12 weeks after implantation. Our findings confirm that functional axonal regeneration fails in conventional long acellular grafts. In this condition we found very few axons in the distal host nerve, and there were marginal signs of sciatic nerve reinnervation in few (2/9) rats. This was accompanied by extensive structural disintegration of the distal graft and abundant retrograde axonal regeneration in the proximal nerve. In contrast, most (8/9) animals receiving nerve repair with ChABC grafts showed sciatic nerve reinnervation by direct nerve pinch testing. Histological examination revealed much better structural preservation and axonal growth throughout the ChABC grafts. Numerous axons were found in all but one (8/9) of the host distal nerves and many of these regenerated axons were myelinated. In addition, the amount of aberrant retrograde axonal growth (originating near the proximal suture line) was markedly reduced by repair with ChABC grafts. Based on these results we conclude that ChABC treatment substantially increases the effective length of acellular nerve grafts.

Chondroitinase applied to peripheral nerve repair averts retrograde axonal regeneration.

Antegrade, target-directed axonal regeneration is the explicit goal of nerve repair. However, aberrant and dysfunctional regrowth is commonly observed as well. At the site of surgical nerve coaptation, axonal sprouts encounter fibrotic connective tissue rich in growth-inhibiting chondroitin sulfate proteoglycan that may contribute to misdirection of axonal regrowth. In the present study, we tested the hypothesis that degradation of chondroitin sulfate proteoglycan by application of chondroitinase at the site of nerve repair can decrease aberrant axonal growth. Adult rats received bilateral sciatic nerve transection and end-to-end repair. One nerve was injected with chondroitinase ABC and the contralateral nerve treated with vehicle alone. After 28 weeks, retrograde axonal regeneration was assessed proximal to the repair by scoring neurofilament-immunopositive axons within the nerve (intrafascicular) and outside the nerve proper (extrafascicular). Intrafascicular retrograde axonal growth was equivalent in both control and chondroitinase treatment conditions. In contrast, chondroitinase treatment caused a pronounced (93%) reduction in extrafascicular retrograde axonal growth. The decrease in axon egress from the nerve was coincident with an increase in antegrade regeneration and improved recovery of motor function. Based on these findings, we conclude that chondroitinase applied at the site of nerve transection repair averts dysfunctional extrafascicular retrograde axonal growth.

Regeneration of axons after nerve transection repair is enhanced by degradation of chondroitin sulfate proteoglycan.

Our past work indicates that growth-inhibiting chondroitin sulfate proteoglycan (CSPG) is abundant in the peripheral nerve sheaths and interstitium. In this study we tested if degradation of CSPG by chondroitinase enhances axonal regeneration through the site of injury after (a) nerve crush and (b) nerve transection and coaptation. Adult rats received the same injury bilaterally to the sciatic nerves and then chondroitinase ABC was injected near the injury site on one side, and the contralateral nerve was injected with vehicle alone. Nerves were examined 2 days after injury in the nerve crush model and 4 days after injury in the nerve transection model. Chondroitinase-dependent neoepitope immunolabeling showed that CSPG was thoroughly degraded around the injury site in the chondroitinase-treated nerves. Axonal regeneration through the injury site and into the distal nerve was assessed by GAP-43 immunolabeling. Axonal regeneration after crush injury was similar in chondroitinase-treated and control nerves. In contrast, axonal regrowth through the coaptation of transected nerves was markedly accelerated and the ingress of axons into the distal segment was increased severalfold in nerves injected with chondroitinase. On the basis of these results we concluded that growth inhibition by CSPG contributes critically to the poor regenerative growth of axons in nerve transection repair. In addition, degradation of CSPG by injection of chondroitinase ABC at the site of nerve repair increased the ingress of axonal sprouts into basal laminae of the distal nerve segment, presumably by enabling more latitude in growth at the interface of coapted nerve. This suggests that chondroitinase application may be used clinically to improve the outcome of primary peripheral nerve repair.