Autologous nerve implantation into denervated monkey skin promotes regeneration of Meissner’s corpuscle

Background

Skin flap transplantation is an efficient method to repair wounds and soft tissue defects in clinical practice, whereas the deprived or reduced sensory function is a serious issue after transplantation [1–6]. Whether the sensation is successfully rebuilt or not will directly affect the surgical efficacy and functional recovery in certain sites such as the hand, mouth and penis [7–10].

Sensory nerve implantation has been used to restore the sensory function of denervated flaps [11,12]. Gilbert et al. [13] routinely coapted the pudendal nerve to the major sensory nerves of the donor free flap, and all patients had an encouraging return of tactile and erogenous sensibility. In the study of Fan et al. [14], the regenerated nerves were noted nearby the implanted nerves 2 months after implantation, and at month 4 the flaps were reinnervated by the sensory nerves. Studies also demonstrated flaps can regenerate abundant nerve endings and peripheric receptors in rabbit models [15,16]. However, the mechanisms underlying the reinnervation following implantation are still unknown. For example, how do the nerve endings and tunicate endings regenerate? How is the feeling restored? [17–19].

Generally, there are 4 cutaneous sensations – touch, cold, warm and pain sensations. Nerve endings classified into free nerve endings and tunicate endings (Meissner’s corpuscles). Meissner’s corpuscle plays an important role in the precise location of sensations. Meissner’s corpuscle is a rapidly adaptive mechanical receptor and responsible for sensitivity to light touch with low threshold. It is involved in the 2-point discrimination, which is very important to the normal function of skin. Following peripheral nerve injury, the regeneration of encapsulated nerve endings is critical for the restoration of sensations [20]. Thus, it is imperative to understand the morphological alternations of denervated sensory endings, especially the Meissner’s corpuscles, following injury [21].

In the present study, a denervated hand model was established in rhesus monkeys, and the sensory ending degeneration following nerve injury was investigated. In addition, the nerve was implanted into the denervated finger flaps, and immunohistochemistry and electron microscopy were employed to observe the morphological and ultrastructural alternations of the degeneration and regeneration of the sensory endings and Meissner’s corpuscles. Electrophysiological testing was performed to determine the type and area of nerve regeneration, aiming to clarify the mechanisms underlying the Meissner’s corpuscle regeneration after nerve implantation.

Material and Methods

ANIMALS:

Nine rhesus monkeys weighing 4–6 kg, regardless of sex, were purchased from the Experimental Animal Center of the 3^rd Military Medical University (Chongqing, China). These monkeys were specific pathogen-free and meet the China National Standard GB14922 (23)-294. This study was carried out in accordance with the guidelines for the care and use of laboratory animals and approved by the ethics committee. Flaps were prepared in a total of 72 2nd-5th fingers. Fingers were randomly divided into 3 groups: normal group (n=5); implantation group (n=5); and non-implantation group (n=5).

SURGICAL PROCEDURES:

The monkeys were anesthetized with ketamine (5 mg/kg, IV). A longitudinal incision was made along the midline in each side of the finger, and 2 digital nerves were separated (Figure 1A). In the implantation group, 1 nerve was cut off at the root of the finger, and the proximal end was fixed backward; the other was directly implanted beneath the dermal layer of the flap skin and its end fixed in the sub-derma of the center tip (Figure 1B). In the non-implantation group, both digital nerves were removed at the root of fingers and both ends were also fixed backward (Figure 1C). Finally, the wounds were carefully closed, and the hands were dressed for 1 or 2 days.

SAMPLE COLLECTION:

The full-thickness skins (1×1 cm) were collected from the finger pulp at the specific time points.

ULTRASTRUCTURAL OBSERVATION UNDER ELECTRON MICROSCOPE:

The samples were fixed in 3% glutaraldehyde and osmic acid for 16 h at 4°C, followed by dehydration in graded ethanol and embedding in epoxy resin. Then, the tissues were consecutively cut into 50 nm-thick sections and double stained by uranyl acetate and sodium citrate. After washing, the sections were examined under a transmission electron microscope.

IMMUNOHISTOCHEMISTRY FOR NEUROFILAMENT IN THE SKIN TISSUES:

The skin tissues were fixed in 4% paraformaldehyde for 12 h, followed by embedding in paraffin. Then, the 30-μm sections were prepared with a frozen slicer. Streptavidin-peroxidase (SP) method was employed to stain the neurofilament in the skins. Briefly, the sections were deparaffinized, and then washed in distilled water according to the standard protocol. After rinsing in PBST (0.01 M PBS, pH 7.4, KH₂PO₄ 0.02%, N₂HPO₄ 0.29%, KCl 0.02%, 0.8% NaCl, 0.05% BSA, Tween-20 0.05%, 0.0015% Triton X-100) (3×5 min), sections were treated with 3% peroxide-methanol at room temperature to inactivate endogenous peroxidase. The following steps were carried out in a moist chamber: (1) Incubation with normal goat serum at room temperature for 20 min; (2) Sections were washed with PBST and treated with rabbit anti-human neurofilament antibody (Abcam, USA) (1:200) for 2 h at 37°C; (3) Washing in PBST (3×5 min); (4) Sections were incubated with goat anti-rabbit IgG (Zhongshan Goldenbridge Company, Beijing, China) for 30 min at 37°C; (5) Rinsing with PBST (3×5 min); (6) Sections were incubated with the SA/HRP at 37°C for 30 min; (7) Rinsing in PBST (3×5 min); (8) Visualization by 3, 3-diaminobenzidin (DAB) (Sigma-Aldrich, St. Louis, MO, USA) treatment at room temperature in dark for 10 min; (9) Sections were washed with distilled water and stained with hematoxylin; (10) After dehydration, mounting was performed with neutral gums. The rabbit anti-monkey neurofilament antibody was replaced by PBS in the negative control group. Finally, the sections were imaged using a laser scanning microscope (Olympus Fluoview FV1000, Japan).

The sections were semi-quantitatively scored as previously described. The immunostaining intensity was classified as: no staining (0), mild staining (1), moderate staining (2), and strong staining (3). Grading was performed according to the percentage of positive cells: <5% (0), 6–25% (1), 26–50% (2), 51–75% (3), and >75% (4). The final score of each section was calculated as follows: score_intensity×score_grade= final score, and the results were expressed as negative (−; 0), weakly positive (+; 1–3), positive (++; 4–7), and strongly positive (+++; 8–12). A minimum of 5 fields were randomly selected from each section for evaluation. The scoring was performed by 2 independent pathologists blind to the study. Any discrepancy was resolved through discussion between the 2 scorers.

NERVE DISSECTION:

Electrophysiological examination was carried out after general anesthesia with ketamine (5 mg/kg, IV) and brachial plexus block at the specific time-points. A 2–3 cm longitudinal incision was made from the root of a finger to the center of the palm, and the proper palmar digital nerve was exposed. The edges of the incision were fixed to form a small bag in which a small quantity of paraffin oil of 37°C was perfused. Then, a single fiber was removed from the nerve trunk with use of a 32X power microscope and a pair of hairspring tweezers. The proximal end of the single nerve fiber was cut and hung to a platinic electrode connected to the VC-11 electrophysiological system (AZ, USA). The alternations of the waves were recorded using its recorder. The detection was repeated at least 30 times for each digital nerve.

STANDARDS FOR FIBER TYPES:

When a responding mechanoreceptor and its afferent single fiber were encountered, the receptive field was located and the absolute response threshold measured with von Frey hair (0.5~100 mN) on its most sensitive point of the receptive field. Then, cold (15°C) and warm (45°C) cotton balls was used as temperature stimuli. The type of this fiber could be identified by the specific adapting behavior. That is to say, the rapidly adapting (RA) fibers respond only to the initiation and discontinuation of 1 or several stimuli, and have no response in continuous skin pressure. However, the slowly adapting (SA) fibers persistently respond to a stimulus for 10 minutes.

DETERMINATION OF CONDUCTION VELOCITY:

The conduction velocity of randomly selected fibers was measured in different groups. The electrical stimuli were administered through 2 thin electrodes penetrating into the skin at the most sensitive point of a single fiber’s receptive field, and the conduction time between a stimulus and the occurrence of action potential was obtained through the oscilloscope. A stable single-action potential was evoked under a synchronic square-wave stimulus that was 115 times higher than the strength of threshold intensity. The width of the output pulse of electrical stimulator was set at 0.1–0.2 m/s. The distance between the stimulus electrodes to the recording electrode is divided by the conduction time and the results represent the conduction velocity of the fiber.

STATISTICAL ANALYSIS:

Data are expressed as mean ±standard deviation (SD). Each experiment was repeated at least 3 times. SPSS version 11.0 statistics software was employed to analyze the differences. Analysis of variance (ANOVA) and T test were used to compare the differences among and between groups, respectively. In addition, chi square test and Pearson correlation analysis were also performed. A value of P<0.05 was considered statistically significant.

Results

ULTRASTRUCTURAL CHANGES AFTER NERVE IMPLANTATION:

Meissner’s corpuscles were observed in the dermal papilla layer. The outer membrane was intact and the intermembranous matrix uniform. The diaphragm was arranged spirally, and no non-myelinated nerve fibers were noted. Visible myelinated nerve fibers and Schwann cells were observed at the end of Meissner’s corpuscles (Figure 2A, B).

At 3 months after denervation, although the outer membrane of the Meissner’s corpuscles was integrated, neural elements began to degenerate, which was mainly characterized by an increased karyoplasmic ratio in Schwann cells at the end of corpuscles, disaggregation or even absence of myelinated fibers, vacuolization of unmyelinated fibers, absence of mitochondria, filaments and microtubules at the axon stump, and formation of cellular tissues.

At 6 months after denervation, the Meissner’s corpuscles were degenerated: (1) No further change was present in the corpuscles beside the blood vessels; and (2) Obvious changes occurred in the corpuscles far from the blood vessels. The morphology was disordered, and had a ruptured membrane, increased internal collagen, decreased and dispersed Schwann cells, increased karyoplasmic ratio, integrated cell membrane and nucleus, and completely collapsing endoneurial tubes (Figure 2C, D).

The Meissner’s corpuscles began to atrophy at 12 months after denervation, and a large amount of collagen fibers were generated. The contents of corpuscle’s Membrane cells and matrix decreased or even disappeared. Schwann cells and their membrane structure disappeared in some Meissner’s corpuscles.

Three months after nerve implantation, the outer membrane was integrated and the intermembranous matrix was uniform. Degenerated nerve fibers were seen at the end of corpuscles, whereas the endoneurial tube was integrated and not collapsing. The number of Schwann cells significantly increased in the corpuscles, accompanied by an increase of cytoplasm, decreased ratio of nucleus to cytoplasm, and increase of circular mitochondrial ridge in the cytoplasm. The regenerated non-myelinated nerve fibers appeared around the Meissner’s corpuscles near to the blood vessels (Figure 2E, F).

Meissner’s corpuscles with simple structure were present at month 6 and were about 1/3~1/2 of normal size. The regenerated corpuscles were located nearby the pre-existing ones. Regenerated axons were found in the center of Meissner’s corpuscles. The Meissner’s corpuscles had no integrated outer membrane structure, and the content of intermembranous collagen fibers was higher than that normal. Newly regenerated nerve fibers were present, accompanied by a sprouting growth inside the corpuscles (Figure 2G).

Further, the structure of Meissner’s corpuscles was improved at 12 months after nerve implantation. One nerve fiber was encapsulated by 2–3 Schwann cells at the end of Meissner’s corpuscles, and the regeneration of medullated nerve fibers was also present. The lamellar structure was regularly arranged, and mitochondria, microfilaments and microtubules were seen at the stump of nerve fibers. Two consecutive Meissner’s corpuscles were re-innervated by 1 nerve tract. The medullated nerve fibers locating at the end of corpuscles became thicker, and the number of layers increased. The morphology of cells on the membrane was integrated, and the arrangement of membranous lamina regular. The majority of Meissner’s corpuscles were mature (Figure 2H, I).

IMMUNOHISTOCHEMISTRY:

In the normal group, a small number of uniform dermal nerve fibers were arranged in orderly fashion. Intact Meissner’s corpuscles were observed in the dermal papilla. The elliptic or spindle-shaped corpuscles with several layer cells in the center were re-innervated by double or multiple nerve axons. The dermal nerve plexus-derived axons continuously gave out branches after entering into the end of corpuscles, and they spirally traversed among the lamellar cells (Figure 3A, B).

In the non-implantation group, intact skin structure was present, accompanied by small aphthae in some tissues. Germinative positive nerve fibers were occasionally observed in the deep dermis, without obvious regeneration of Meissner’s corpuscles (Figure 3C).

The regenerated nerve fibers in the derma significantly increased at 3 months after nerve implantation. The nerve fibers regenerated in a pattern of single fibers and small clusters. Axons were observed in the dermal papilla, accompanied by the regeneration of Meissner’s corpuscles. The corpuscles were re-innervated by single axons with less axon component but more collagen fibers (Figure 3D).

The nerve fibers in the derma had regenerated extensively at 6 months after implantation. The number of nerve fibers was higher than that normal and the nerve fibers were distributed intensively. The number of regenerated nerve fiber clusters was increased and the proportion of myelinated fiber was elevated. However, the growth rate of nerve fibers markedly attenuated when compared with that at month 3. The regenerated Meissner’s corpuscles significantly increased. These corpuscles had more axon component and fewer collagen fibers re-innervated by multiple axons (Figure 3E, F).

The number of regenerated nerve fibers was maintained at 12 months after nerve implantation (Figure 4A). The regenerated nerve fiber clusters were arranged in order. The proportion of myelinated fibers increased and that of nonmyelinated fibers decreased. The number of regenerated Meissner’s corpuscles dramatically increased and some were close to the normal corpuscles (Figure 4B). They were mainly re-innervated by multiple axons in a typical helix pattern.

NERVE TRUNK IMPLANTATION INCREASES THE NUMBER OF AFFERENT FIBERS OF MECHANORECEPTOR:

In the non-implantation group, the responsive mechanoreceptors appeared only at the region where the proximal end of transected nerve was sutured in the distal palm; very few were found at the proximal palmar nerve of the finger and none were noted beyond the level of the proximal knuckle.

In the normal group and the implantation groups, receptive fields of the re-innervated mechanoreceptors were located regularly from the root to the tip of a finger. To exclude the interference by the growth of regenerated proximal fibers, only a mechanoreceptor whose receptive field was located in the middle and distal phalanx zones was treated as being re-innervated, ensuring that the mechanoreceptor was re-innervated only by the implanted nerve. According to the above criteria, the number of re-innervated mechanoreceptors following nerve implantation was determined and expressed as a percentage. The proportion of re-innervated mechanoreceptors rapidly reached up to 47.7% 3 months after nerve implantation. Thereafter, this proportion gradually increased at month 6 and 12. However, the level of re-innervated mechanoreceptors in the implantation group was still lower than that in the normal group 12 months after implantation (Figure 5A).

PROPORTIONS OF REGENERATED FAST ADAPTIVE FIBER (RAFS) AND SLOW ADAPTIVE FIBER (SAFS) MECHANORECEPTORS: The proportion of re-innervated RAFs mechanoreceptors in the implantation group was markedly higher than that in the normal group at months 3 and 6. However, there was no significant difference in the RAFs and SAFs mechanoreceptors 12 months after nerve implantation between the normal group and implantation group (Figure 5B, C).

ALTERNATIONS OF THE THRESHOLD OF RE-INNERVATED MECHANORECEPTORS: The mean threshold of RAFs and SAFs were significantly higher in the implantation group than those in the normal group 3 months after nerve implantation. The threshold remained stable 6–12 months after implantation. The mean RAFs threshold reached the near normal level and the SAFs threshold was slightly higher than the normal level at months 6 and 12 (Figure 6).

Discussion

In the present study we successfully prepared the denervated finger flap in monkeys, and our results revealed that nerve trunk implantation into the denervated flap could promote the nerve regeneration, skin re-innervation and regeneration of Meissner’s corpuscles.

Our results showed the Meissner’s corpuscles in the denervated hairless skin began to degenerate. The axons ruptured and disappeared, and the endoneurial tubes began to collapse. The theca cells were disrupted and disappeared at month 12, and the Meissner’s corpuscles were rich in collagens, but still relatively small [21].

Generally, the degeneration of Meissner’s corpuscles is a relatively long process. The nerve degeneration occurred earlier and the development of non-nerve degeneration was prolonged. Interestingly, the nerve implantation group displayed a better nerve restoration when compared with the non-implantation group. Two mechanisms – growth along the endoneurial tube and direct axon growth – might be responsible for the regeneration of Meissner’s corpuscles. The process of regenerated Meissner’s corpuscle development includes the growth of axons inside the end of corpuscles along the endoneurial tube or repeated sprouting, being packaged by theca cells, and development into the corpuscle body [22,23]. At the early stage, the axons at the terminal part of Meissner’s corpuscles were un-myelinated. The myelinated axons were present at month 8, and thickening of medulla sheath and increases of layers were obvious at month 12. The morphology of theca cells was intact and the membranous lamina was arranged regularly, suggesting the Meissner’s corpuscles were largely matured. These results strongly suggest that Meissner’s corpuscle is a basic structure for sensation. Studies on the degeneration, regeneration, pattern, pathway, and regeneration degree of Meissner’s corpuscles will be helpful to further illuminate the mechanism underlying the therapeutic effects of sensory nerve implantation [24,25].

The structure of the implanted skin in the present study was between full-thickness skin graft and transfer of skin flap, which is similar to the structure in the regeneration of skin sensory receptors in clinical practice. Currently, the regeneration of implanted skins with sensation is still controversial [26,27]. The regeneration of implanted nerves is determined by the original skin characteristics, but not the skin receptive field [28]. However, a large number of regenerated Meissner’s corpuscles were observed in the implanted palm skin, suggesting that implanted skin can regenerate tunicate receptors [29]. Implanted skin has been found to have both a large number of regenerated nerve endings and considerable regenerated Meissner’s corpuscles and epithelium-axon complex. The nerve fibers regenerated and their number finally exceeded that in the normal skin. The process of the development from non-myelinated fibers to myelinated fibers is of great importance to: (1) establish the sensation pathway and provide imprecise sensation as free endings between epidermis and dermis; and (2) provide more opportunities for axons to grow into appropriate terminal organs in a pattern of excessive regeneration. In addition, it interacted with degenerated Meissner’s corpuscle and enabled the corpuscle to regenerate, achieving a favorable sensory restoration [30,31].

The results of this study and our previous studies [18,19] show that sensory nerve implantation can achieve good sense reestablishment in the middle- and long-term. Some mechanisms may be responsible for this protective effect: (1) The implanted sensory nerves are mainly thin and have small nerve tracts, and the axonotmesis is far away from the center and near the end. Axonal transection had little effect on the perikaryon, which is beneficial for the nerve regeneration following implantation. (2) The regenerated axons sprouted and inhibition was absent during the process of growth due to anastomosis scar. (3) The regenerated axons may interact with the original degenerated corpuscles or form free endings after growing in or under the derma for a relatively short distance. This shortens the time in degeneration and reduces the degree of degeneration, which are helpful for the reestablishment and restoration of Meissner’s corpuscles [32,33].

Conclusions

In conclusion, in the present study we successfully prepared denervated flaps in the fingers of monkeys. Not only a large number of regenerated nerve endings, but also considerable regenerated Meissner’s corpuscles were found 12 months after nerve implantation. The number of nerve fibers was higher than in the normal group, and the type of nerve fibers also increased. The gradual regeneration of Meissner’s corpuscles suggests a favorable recovery of sensation at the middle- and long-term neurotization [34,35].