Limited regeneration in long acellular nerve allografts is associated with increased Schwann cell senescence

Abstract

Repair of large nerve defects with acellular nerve allografts (ANAs) is an appealing alternative to autografting and allotransplantation. ANAs have been shown to be similar to autografts in supporting axonal regeneration across short gaps, but fail in larger defects due to a poorly-understood mechanism. ANAs depend on proliferating Schwann cells (SCs) from host tissue to support axonal regeneration. Populating longer ANAs places a greater proliferative demand on host SCs that may stress host SCs, resulting in senescence. In this study, we investigated axonal regeneration across increasing isograft and ANA lengths. We also evaluated the presence of senescent SCs within both graft types. A sciatic nerve graft model in rats was used to evaluate regeneration across increasing isograft (~autograft) and ANA lengths (20, 40, and 60 mm). Axonal regeneration and functional recovery decreased with increased graft length and the performance of the isograft was superior to ANAs at all lengths. Transgenic Thy1-GFP rats and qRT-PCR demonstrated that failure of the regenerating axonal front in ANAs was associated with increased levels of senescence related markers in the graft (senescence associated β-galactosidase, p16(INK4A), and IL6). Lastly, electron microscopy (EM) was used to qualitatively assess senescence-associated changes in chromatin of SCs in each graft type. EM demonstrated an increase in the presence of SCs with abnormal chromatin in isografts and ANAs of increasing graft length. These results are the first to suggest that SC senescence plays a role in limited axonal regeneration across nerve grafts of increasing gap lengths.

Keywords: 4′,6-diamidino-2-phenylindole; ANAs; Acellular nerve allograft; Cellular senescence; DAPI; EDL; GFP; Nerve autograft; Nerve grafting; Peripheral nerve; SCs; Schwann cell senescence; Schwann cells; SenScs; acellularized nerve allografts; extensor digitorum longus; green fluorescent protein; senescent Schwann cells.

Fig. 1

Evaluation of nerve regeneration in long graft model in rat. A) Two sciatic nerve grafts (lengths of >30 mm) were harvested from a single donor rat. The two nerve pieces were coapted together in a proximal–distal end to end fashion to form a graft of up to 60 mm. The coapted donor was then trimmed to the desired length (40 or 60 mm) for nerve interposition and implanted in a “pocket” under the skin. (P indicates proximal. D indicates distal. Arrows indicate suture lines). Histomorphometric analysis of regenerating nerve fibers demonstrated decreased axonal regeneration with increased graft lengths in both graft groups. The total number of myelinated nerve fibers was quantified at 10 weeks (B) and 20 weeks (C) after reconstruction. At both time points, isografts (ISO) demonstrated superior regeneration in comparison to ANAs at all lengths. D) Representative histological sections of regenerating nerve 5 mm into the distal nerve stump at 400× magnification were taken. Sections acquired from 40 mm and 60 mm nerve isografts showed robust axonal regeneration, numerous myelinated fibers, and mature nerve architecture. The section acquired from 40 mm ANA also demonstrated some axonal regeneration. In contrast, the section acquired from 60 mm ANA graft demonstrated no healthy, myelinated axon. E, F) Assessment of recovery of EDL muscle mass demonstrated that there was a positive correlation between muscle atrophy and graft length for both isograft and ANA graft. E) EDL muscle recovery (the ratio of experimental/contralateral) after 10 weeks demonstrated superior recovery for animals reconstructed with isografts. F) Similarly, recovery of EDL muscle mass after 20 weeks demonstrated increased recovery in isograft treated groups. In contrast, groups treated with ANA exhibited increased atrophy over the time period between 10 and 20 weeks. G) Functional reinnervation after 20 weeks measured by evoked tetanic muscle force in the EDL demonstrated no significant reinnervation in the 40 mm and 60 mm ANA groups. In contrast the isograft treated groups at both lengths exhibited significantly increased tetanic muscle force. ISO: isograft. ANA: acellular nerve allograft. Tetanic SpF: Specific isometric tetanic force. *: p < 0.05, **: p < 0.01, ***: p < 0.001. Error bars present the mean ± standard deviation (SD).

Fig. 2

Axonal regeneration in Thy1-GFP rats. Thy1-GFP rats express green fluorescent protein (GFP) in their axons allowing for visualization of the regenerated axons in grafts after 10 weeks (A). A strong inverse relationship between the axonal regeneration and the length of ANA was observed: Both 20 mm isografts (B) and ANAs (E) were able to support axonal regeneration through the length of the graft. When the graft length was increased to 40 mm, axonal regeneration was hindered in ANAs (F) but not isografts (C); this difference in extent of regeneration was even more pronounced at 60 mm (D & G). Of note, the axons regenerated a shorter distance in the 60 mm ANA (G) than in the 40 mm ANA (F). All images were obtained at 6.3× magnification. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Immunohistochemical staining for markers of senescence. Long nerve grafts (isografts n = 9, ANAs n = 9) were stained for senescence marker, ß-galactosidase (blue), and counter-stained for nuclei with nuclear fast red stain (red). A) A representative image of ß-galactosidase stained isograft and ANA are shown. Each tissue section was analyzed in six regions along the length of the graft to quantify the presence of senescent cells. B) A manual count of the average ratio of nuclei staining positively for ß-galactosidase at each of 6 locations along 60 mm isografts and ANAs was performed. C) Long nerve grafts (isografts n = 4, ANAs n = 4) were also immunostained for markers of SCs (S100) and senescence (p16). The median intensity of fluorescence from each marker was quantified. The results of p16^INK4A evaluation are shown in (C). D) A representative image of an ANA and isograft is shown, with areas staining positively for p16^INK4A shown in green and S100 in red. Blue areas indicate presence of cellular nuclei, which are stained with DAPI. E) A representative transverse section within the graft of an ANA was immunostained for senescent and SCs specific markers (20× image, scale bar is 50 μm, blue staining is DAPI for cell nuclei). The same tissue section was stained for the senescence marker p16^INK4A (green left panel) and the SC specific marker S100 (red left panel). F) Colocalization of both markers in areas staining positively for both p16^INK4A (green) and S100 (red) appears yellow, and indicates the expression of the senescent marker p16^INK4A within SCs. (ISO: isograft. ANA: acellular nerve allograft. *: p < 0.05, **: p < 0.01, ***: p < 0.001. Error bars present the mean ± standard deviation (SD)). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

Quantification of senescent gene expression in long nerve grafts. The graft and portions of the proximal and distal nerve stumps were harvested after 10 weeks from each experimental group (n = 4). A) The tissue was sectioned into five segments. B) Each segment (proximal host, proximal graft, middle graft, distal graft, and distal host) was processed to quantify the expression of senescent (p16^INK4A (p16), p53, and IL6), and Schwann cell (S100, p75 (Ngfr)) related mRNA. (ISO: isograft. ANA: acellular nerve allograft. Dotted line indicates 2 fold expression. Error bars present the mean ± standard deviation (SD)).

Fig. 5

Chromatin reorganization in senescent cells. DAPI staining revealed clumping of DNA in SC nuclei in the distal ANA (A), consistent with the presence of senescence-associated heterochromatin foci. Closer examination of nuclei of SCs for changes in the chromatin within a representative 20 (B), 40 (C), 60 (D) mm ANA and isografts was performed under electron microscopy. The analysis revealed a progression of clumping of chromatin with central involutions in the 40 and 60 mm ANA (orange outlines and arrowheads). These augmented nuclei were found in the region of stalled axonal front and persisted throughout the distal graft. The DNA clumping starkly differed from the uniform peripheral appearance of chromatin in short graft ANA and the normal proximal host (gray outline). Similar changes were noted in the isograft but at a more distal location and only in the 60 mm graft length. All EM images were obtained at 10,000× magnification. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)