Severe nerve injuries and neurolytic procedures resulting in a loss of substance leave gaps between nerve stumps and preclude direct epineural repair. Utilizing autologous grafts to bridge the gap requires the sacrifice of a nerve of lesser functional significance . While this method has resulted in the best regenerative results to date, it necessitates a second surgical step, sacrifices donor nerve function, and depends upon a finite supply of potential donor nerves [2, 3]. The development of a biosynthetic nerve conduit would permit repair of peripheral nerve defects too large to be repaired by autologous grafts while circumventing the tactical shortcomings described above. Yet, despite considerable effort, current conduits perform inferiorly when compared to autografts [2, 3, 4].
Even when axonal regrowth is facilitated, appropriate axonal targeting must still occur to restore original innervation and permit physiologic recovery. While the central and peripheral nervous systems (CNS and PNS, respectively) respond very differently to injury, neither one can effectively produce both adequate regeneration and appropriate targeting [5, 6, 7]. The mammalian olfactory system is a notable exception.
Mammalian olfactory systems are unique in that their axons must constantly regenerate and appropriately retarget in order to overcome the constant damage and repair experienced within the hostile environment of the nasal cavity [8, 9]. This capacity for regeneration is facilitated by the olfactory ensheathing cell (OEC) – a specialized Schwann cell whose properties include modulation of astrocytes/fibrocytes to minimize scarring , promotion of neoangiogenesis , and release of neurotrophic factors . The past decade has seen wide use of OEC engrafting and transplantation in both CNS and PNS [4, 13, 14]. Autologous OEC transplantation has improved the efficacy of nerve conduits in peripheral nerve regeneration [2, 3, 4] yet such enhanced conduits remain inferior to autologous nerve grafts [4, 15, 16]. Perhaps this is because OECs rely on recruitment of remaining axons at the site of injury .
Vomeronasal organ (VNO) is a bilaterally paired pseudostratified sensory epithelium for accessory olfaction located in the nasal septum of most vertebrates . Although its stem cell population has not been definitively characterized, VNO remains neurogenic throughout the life of an individual and, like the main olfactory system, exhibits targeted synapse establishment throughout adulthood, even following complete axotomy [19, 20]. VNO has been transplanted into brain and spinal cord [21, 22], where it offers the combined effects of both a stem cell graft and an OEC graft. Its OECs promote axonal regeneration while its stem cells produce new neurons and axons at the site of injury [2, 20, 21, 22]. The present study is the first transplantation of VNO into the peripheral nervous system.
Because intact VNO tissue transplants in CNS both promote axonal regeneration and provide a new population of axons for recruitment , we hypothesize that in PNS they have the potential to facilitate nerve regeneration in ways previously unobserved. If optimized, such a discovery would be the first step in the creation of a biosynthetic product which would provide an alternative to autologous nerve grafting in bridging neural gaps.
Donor animals were seven neonatal Sprague-Dawley rats (Harlan Laboratories, Indianapolis, IN, USA). Recipient animals were 21 adult female Sprague-Dawley rats (Harlan Laboratories, Indianapolis, IN, USA) weighing between 180 and 500 g. Rats were housed in clear plastic cages with stainless steel tops and maintained on a 12-hour light and dark cycle with the temperature and relative humidity of the animal room controlled at 21–23 C and 55–65%, respectively. Rats were allowed free access to food and water until one hour prior to the experiments. All procedures described below were approved by the Institutional Animal Care and Use Committee (Protocol # A004-07-01).
Thirty donor rats were anesthetized with isoflurane vaporized in oxygen in a clear box and decapitated. VNO was obtained for transplantation via mid-transverse incision of the hard palate, eversion of the nasal septum, and sharp bilateral dissection of the vomeronasal cartilage. VNO epithelium was cut into fragments approximately 1 mm3 and placed in Ham’s F-10 media (Bio Whittaker, Walkersville, Maryland) on ice.
Thirty recipient rats were anesthetized using inhaled isoflurane vaporized in oxygen. Rats were placed in a clear box into which vaporized isoflurane was delivered in oxygen at an induction concentration of 3–4%. Following induction, anesthesia was maintained by means of mask-delivery of isoflurane in a Bain circuit at a concentration of 0.5–2.5% for the remainder of each experiment.
After clipping of the fur, the right sciatic nerve of 20 recipient rats was exposed by a craniolateral approach which exploits the interval between the biceps femoris and the superficial gluteal muscles. Under an operating microscope, the sciatic nerve was transected 10 mm distal to the sciatic notch and a 10 mm segment of nerve was sharply excised. Both nerve ends were then inserted a distance of 1 mm into a 2.0 mm diameter commercially available collaen nerve conduit of 10 mm length (NeuraGen, Integra Life Sciences, Plainsboro, NJ, USA), resulting in an inter-stump gap of 8 mm. Both nerve ends were attached to the tube by means of 7–0 Nylon (Ethicon, Somerville, NJ, USA) epineural sutures to prevent tension. In 10 rats, the inter-stump space was filled with saline only (conduit only or “CO” group). In 10 rats, the inter-stump space was filled carefully with fresh donor VNO (“VNO” group). In an additional 10 rats, the sciatic nerve was exposed and transected as described above, then repaired by means of three 7–0 Nylon epineural sutures (epineural repair or “ER” group). After irrigation, wound closure was accomplished in two layers using 4–0 PDS and 4–0 Chromic gut (Ethicon, Somerville, NJ, USA).
At 14 weeks postoperatively, all animals underwent walking track analysis as described by Inserra et al. (1998). Rear paws were dipped in ink and animals walked upon absorbent paper (GMP Chem Tech, Anaheim, CA, USA). Morphometric analysis of the prints for toe spread and paw length was made manually. SFI was calculated using the formula described by Inserra et al. (1998) where a score of zero represents equal function to the contralateral limb and -100 represents total loss of sciatic nerve function:
Operated sciatic nerve was harvested from the sciatic notch to the stifle and post-fixed overnight by immersion in 4% PFA. After embedding, 1-um thick axial specimens were sectioned and stained with toluidine blue. Digital photomicrographs were acquired on a Zeiss microscope (Carl Zeiss, Jena, Germany) and imported into ImageJ image processing software (National Institutes of Health, Bethesda, MD) for blinded analysis. Total nerve area was measured for each nerve on low-power magnification. Each axial section was divided into four quadrants, and two to three high-power fields (1000×) from each quadrant were evaluated for myelinated axon number and average axon diameter. Unmyelinated axons and axons smaller than a predetermined diameter were not counted. Depending on the diameter of the nerve being examined, this counting method encompassed 30% to 100% of the total nerve area. Axon number was divided by the area sampled to calculate average myelinated axon density.
Data were analyzed using SAS 9.1 (SAS Institute, Cary, NC). Overall differences among medians between groups were determined using the Kruskal-Wallis test with a 95% confidence interval. Direct comparisons between groups were calculated based on a Wilcoxon rank sum test of difference between medians with a 95% confidence interval. Differences between groups were considered significant for p < 0.05 for all tests. Data are expressed as a mean with standard deviation, number of animals, and corresponding p-values indicated.
All animals survived treatment without any serious surgical complications. Animals were examined daily for the first week postoperatively and once or twice weekly afterwards by a single veterinarian. All animals suffered decubitus ulcers in the right foot and digital pads (Figures 8 and 9). There was marked atrophy in the tibialis anterior and peroneal muscles of the right rear limbs. Marked clawing of the digits consistent with intrinsic foot muscle contracture was noted in all animals (Figures 8 and 9). Foot ulceration, muscle tone and bulk, and foot posturing were all noted to improve postoperatively. At sacrifice, all sciatic nerves were in continuity. The collagen conduit material showed variable amounts of absorption upon sacrifice but was not associated with any detectable inflammatory host response.
Mean sciatic functional index (SFI) was measured in each of the three groups at 14 weeks postoperatively (Figure 10). Mean SFI was highest in the epineural repair (ER) group (–14.56), followed by the VNO-enhanced (VNOE) group (–17.66), followed by the saline-filled conduit-only (CO) group (–48.09) (Figure 3). Mean SFI in the VNOE group was significantly higher than in the CO group (p = 0.006). Mean SFI in the ER group was also significantly higher than in the CO group (p = 0.004). Mean SFI was statistically equivalent between the VNOE and ER groups (p = 0.338) (Table 1).
|Variable||Statistic||ER||CO||VNOE||Overall P-value*||ER vs CO P-value**||ER vs VNOE P-value**||CO vs VNOE P-value**|
|Mean (SD)||–14.56 (13.43)||–48.09 (11.06)||–17.66 (12.79)||0.004||0.004||0.338||0.006|
|Mean (SD)||3981.39 (3593.27)||3706.46 (2740.81)||3443.98 (2091.32)||0.972||0.949||0.701||1.000|
|Total Nerve Area||N||7||7||7|
|Mean (SD)||566140 (484080.78)||532165.43 (380582.33)||449082.29 (373180.39)||0.948||0.848||0.898||0.749|
|Mean (SD)||9.15 (9.34)||3.59 (2.33)||9.67 (4.92)||0.023||0.048||0.306||0.013|
Analysis of total nerve area showed no significant differences between any groups. There was a significant treatment effect for axon density (Table 1). Nerves in the VNOE group had a higher mean axon density (p = 0.013) than nerves in the CO group. Nerves in the ER group also had a higher mean axon density (p = 0.04) than nerves in the CO group. Mean axon density was statistically equivalent between nerves in the VNOE and ER groups (p = 0.306) (Figure 11). A histogram plot showing the relationship between sciatic functional index (SFI) and axon density is shown in Figure 12.
Segmental nerve defects remain a challenging problem in peripheral nerve reconstruction. Autologous nerve grafting represents the current gold standard in repair of segmental nerve defects but requires sacrifice of another peripheral nerve. Nerve conduits using biological (e.g. vein wrapping) and non-biological (e.g. silicon, polyglycolic acid, or collagen tubes) materials have been devised [2, 3, 4]. Various means of conduit enhancement have been attempted in the hope of creating a biosynthetic alternative to autologous nerve grafting, including modifying conduits by the addition of neurotrophic factors such as laminin, peripheral neural elements such as Schwann cells, central neural elements such as olfactory ensheathing glia, and a variety of neural progenitor cells [2, 4, 16, 23, 24, 25]. With regard to the latter category, conduit enhancement using neural progenitor cells has proven controversial, costly, and technically challenging. To date, none of these alternatives have shown results equivalent to autologous nerve grafting.
This study introduces the use of an intact neuroepithelium—vomeronasal organ (VNO)—as a novel tissue for nerve conduit enhancement. Although the exact mechanisms by which olfactory neuroepithelial tissues regenerate are not entirely clear, they represent a pluripotent neural tissue which is robust and relatively easy to harvest. The data presented here demonstrated the effectiveness of VNO in promoting sciatic functional recovery in rats when compared to a standard, non-enhanced collagen nerve conduit. VNO maintains its capacity for both neural regeneration and appropriate neural targeting throughout adult life due to two key features; the presence of olfactory ensheathing cells which permit axonal elongation, and a stem cell population which permits neuronal replacement . Because the exact nature of VNO function is not well understood and the individual components have not been separated in culture, we sought to investigate the potential utility of VNO in peripheral nerve regeneration by means of whole tissue transplantation into a site of segmental nerve loss.
In rat models of segmental sciatic nerve loss, defects smaller than 10 mm are generally considered to be amenable to repair via bridging with a tubular conduit [25, 27]. For our first control group we selected a 10 mm defect (with an 8 mm residual gap after positioning) because it would be expected to regenerate following repair with a standard conduit only (CO). We chose a second control group consisting of simple sciatic nerve transection and direct epineural repair (ER), which could be expected to have greater sciatic functional recovery than any group with segmental loss. The high survival rate of animals, the absence of gross inflammation, and the presence of nerves in continuity at sacrifice indicate that this model is technically viable and will be easy to replicate for future investigations.
One significant outcome in the present investigation was that mean sciatic functional index (SFI) in rats receiving nerve repair via a VNO-enhanced conduit (VNOE) was statistically identical to rats receiving direct epineural repair (ER). Both of these techniques resulted in superior sciatic nerve function compared to rats receiving nerve repair via a standard conduit without VNO enrichment (CO). It is possible that these results related to the effects of VNO on the provisional matrix formed following nerve injury, which is initially composed primarily of fibrin . Regeneration is impaired if the matrix is absent , disorganized , or contains excessive amounts of collagen or laminin . Perhaps the presence of VNO within the nerve conduit creates a more favorable milieu for the stages of nerve regeneration in which Schwann cells and macrophages migrate into the provisional matrix.
Another significant outcome in the present investigation was the presence of a higher axon density in regenerated nerves treated with a VNO-enhanced conduit (VNOE) than in regenerated nerves treated with an empty conduit (CO). This effect may have been related to the axonal growth phase of repair which occurs 2 to 4 weeks postoperatively and coincides with degradation of the provisional matrix. In a rodent model, an investigation of VNO transplantation into the brain demonstrated that many of the axons crossing the glial scar caused by the trauma of transplantation are derived from the transplanted VNO [21, 22]. If VNO-derived axons are indeed present within conduits and available for synaptic targeting as the neuromuscular unit regenerates then this would explain both the higher axon density as well as the improved functional scores. The next investigation will test this hypothesis by using transgenic green fluorescent protein-expressing animals as donors so that host-derived and VNO donor-derived elements may be distinguished from one another.
A third proposed effect of VNO tissue within the site of nerve regeneration relates to target-derived trophic feedback. The process of axonal sprout formation and regression has been well-described [30, 31, 32]. Regenerating axons produce up to 3 to 4 sprouts per axon which cross the site of a peripheral nerve segmental defect . Axonal sprouts which reach endoneurial tubes in the distal segment may regenerate to the target sensory receptor or neuromuscular junction. Axonal sprouts which do not reach target organs undergo resorption due to the lack of a trophic signal [31, 32]. The presence of VNO may influence the trophic effect on regenerating axons, either by providing a nascent source of trophic signaling (e.g. as regenerating axons reach the bipolar neurons from the VNO) or by providing trophic factors that stimulate and sustain axons before they regenerate to the distal nerve segment. Further testing of this hypothesis will depend upon expanding our understanding of how VNO regenerates its own axons in hostile environments.
A limitation of this study is that it did not evaluate the ability of regenerated nerves in this study to regain appropriate CNS targeting. Although it seems a reasonable presumption, SFI does not necessarily correlate with the re-establishment of functional synapses. Future studies should include compound motor action potential measurement and retrograde axonal tracing from muscle distal to the nerve defect back to the central nervous system. SFI and axonal quantification also did not address the tissue of origin of regenerated axons. This study also lacked serial SFI measurement over time. Because of the adverse effects of a sciatic nerve defect model on subject animals, IACUC approval for this project was dependent upon a finite postoperative survival time. To obtain serial functional and electrical data over multiple different time-points, a different animal care and use framework or an animal model with less postoperative morbidity would have to be established.
In summary, the present study suggests that a sciatic nerve reconstruction with a nerve guidance conduit enhanced with VNO offers functional and histomorphometric advantages to reconstruction with a standard conduit. Although these results are encouraging, many questions remain. Future studies should investigate axonal origin (VNO donor versus host), axonal targeting, compound motor action potential evolution, and recovery of sciatic function over multiple time points.
Funding was provided by the Orthopaedic Research and Education Foundation through a Resident Education Grant.
The authors have no competing interests to declare.
William C. Eward, MD, DVM.
This is a non-commissioned paper that has undergone external peer review according to journal policy.
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