Brachial plexus lesion results in loss of motor and sensory function, being more harmful in the neonate. Therefore, this study evaluated neuroprotection and regeneration after neonatal peripheral nerve coaptation with fibrin sealant. Thus, P2 neonatal Lewis rats were divided into three groups: AX: sciatic nerve axotomy (SNA) without treatment; AX+FS: SNA followed by end-to-end coaptation with fibrin sealant derived from snake venom; AX+CFS: SNA followed by end-to-end coaptation with commercial fibrin sealant. Results were analyzed 4, 8, and 12 weeks after lesion. Astrogliosis, microglial reaction, and synapse preservation were evaluated by immunohistochemistry. Neuronal survival, axonal regeneration, and ultrastructural changes at ventral spinal cord were also investigated. Sensory-motor recovery was behaviorally studied. Coaptation preserved synaptic covering on lesioned motoneurons and led to neuronal survival. Reactive gliosis and microglial reaction decreased in the same groups (AX+FS, AX+CFS) at 4 weeks. Regarding axonal regeneration, coaptation allowed recovery of greater number of myelinated fibers, with improved morphometric parameters. Preservation of inhibitory synaptic terminals was accompanied by significant improvement in the motor as well as in the nociceptive recovery. Overall, the present data suggest that acute repair of neonatal peripheral nerves with fibrin sealant results in neuroprotection and regeneration of motor and sensory axons.
Upper and lower limb innervation is greatly affected by brachial and lumbosacral plexus lesion, leading to loss of motor and sensory function [
Experimentally, a well-accepted model to mimic axotomy injury retrograde repercussion to spinal neurons is the neonatal peripheral nerve axotomy [
To date, neonatal peripheral nerve repair following neurotmesis is largely limited due to technical drawbacks. In fact, to perform end-to-end coaptation by epineural/perineural suturing in the newborn represents a major challenge. On the contrary, reconnection of transected stumps by using biocompatible sealants may significantly facilitate the process, as well as taking the advantage of employing the adhesive as scaffold to engraft stem cells, as well as neurotrophic substances, to the injury site.
Fibrin sealant has been applied in neurosurgery for decades, being effective and biocompatible, with no side effects to the nervous system microenvironment [
Although anatomical repair of spinal roots and other lesioned plexus components constitute the primary approach, additional strategies are necessary to enhance neuroprotection and to improve the regenerative response of severed neurons. This may be achieved by stem cell therapy that has shown particularly important positive effects following nervous system injury [
The present work investigated the viability and efficiency of neonatal sciatic nerve end-to-end coaptation, performed with the application of fibrin sealant. We show that both commercial and customized nonhuman fibrin adhesives perform equally well and allow motor and sensory recovery up to 12 weeks after injury.
Nerve stumps coaptation was performed by using either a commercially available fibrin sealant-Tissucol (Baxter®, Vienna, Austria) or a patented fibrin sealant derived from snake venom, kindly supplied by the Center for the Study of Venoms and Venomous Animals (CEVAP) of UNESP (patent registration numbers BR1020140114327 and BR1020140114360). At the time of use, the components were previously thawed, reconstituted, mixed, and applied [
Two-day-old (P2) neonatal Lewis rats were anesthetized by hypothermia and subjected to unilateral sciatic nerve transection at mid-thigh level. Animals were divided into three groups: axotomy alone (AX,
Both fibrin sealants used herein are composed of three separate solutions: (1) fibrinogen, (2) calcium chloride, and (3) thrombin or thrombin-like (in the case of the serine protease derived from snake venom). During surgical repair, the first two components were applied and the proximal and distal stumps were approximated in an end-to-end fashion. The third sealant component was then added for polymerization. The sciatic nerve was gently shifted to assure the stability of the coaptation and to evaluate the success of the repair.
The Institutional Committee for Ethics in Animal (CEUA/UNICAMP) and The Center of Experimental and Ethical Animal (CEEA/UNESP) approved all experiments (proc. number 2593-1 and 904-2011, resp.), which were performed in accordance with the guidelines of the NIH and the Brazilian College for Animal Experimentation. The animals were housed using a 12 h light/dark cycle and controlled temperature (23°C), with free access to food and water. Pups were maintained with the mother until 21 days old, being weaned afterwards.
Animals were anaesthetized with an overdose of Kensol (Xylasine, Köning, Argentina, 10 mg/kg) and Vetaset (Ketamine, Fort Dodge, USA, 50 mg/kg), and the vascular system was rinsed by transcardial perfusion with phosphate buffer 0.1 M (pH 7.4). For neuronal survival counting and the immunohistochemical evaluation, subjects (
Counting of motoneurons survival was performed on spinal cord sections (
The motoneurons were identified based on their morphology and location in the ventral horn (dorsolateral lamina IX). Only cells with visible nucleus were counted for every twentieth section (totalizing an interval of 240
The percentage of surviving cells was analyzed by the ratio of absolute numbers of motoneurons, counted per section, on the lesioned and nonlesioned sides, respectively, and multiplying the result by 100. Abercrombie’s formula [
Slides containing transverse spinal cord sections were incubated for 45 min in a 3% BSA solution followed by incubation with the following primary antibodies: mouse anti-synaptophysin (Dako, Glostrup, Denmark 1 : 200) rabbit anti-GFAP (Abcam, 1 : 1500), and rabbit anti-Iba1 (Waco, 1 : 700). The primary antibodies were diluted in a solution containing 1% BSA (bovine serum albumin) and 2% triton in PB 0.1 M (phosphate buffer). All sections were incubated for three hours in a moist chamber at room temperature. After rinsing in PB, the sections were incubated with a Cy3-conjugated secondary antiserum (1 : 250, Jackson Immunoresearch, West Grove, PA, USA) for 45 min in a moist chamber at room temperature. The sections were then rinsed in PB, mounted in a mixture of glycerol/PB (3 : 1), and observed with a Leica DM5500B microscope coupled with a Leica DFC345 FX camera.
For quantitative measurements, three representative images of the ipsilateral and contralateral ventral horn were captured from each animal for all experimental groups (
Morphometry, regenerated axon area, and counting analyses were performed by sampling at least 30% of each nerve cross-section (magnification of 1,000x) using a bright field microscope (
Lumbar spinal cords (
The terminals were typed as F-type (flattened synaptic vesicles or flattened and spherical vesicles that contain glycine/gamma-aminobutyric acid (GABA) as neurotransmitter), S-type (spherical synaptic vesicles that contain glutamate as the neurotransmitter), or C-type (presence of a subsynaptic cistern and acetylcholine as the neurotransmitter), according to the procedure described by Conradi in 1969 [
Motor function was analyzed using the peroneal functional index (PFI) by the walking track test (CatWalk system, Noldus Inc., The Netherlands;
The electronic von Frey test (Insight Instruments Inc., Ribeirão Preto, SP, Brazil) was utilized to evaluate sensory function recovery (
The data are presented as mean
Neuronal survival was investigated as the ipsi/contralateral ratio of motoneurons present at ventral horn lamina IX. After 4, 8, and 12 weeks, a severe degeneration motoneuron was observed in the AX group. Coaptation groups performed equally well and presented statistically significant rescue of axotomized motoneurons (Figure
Nissl-stained spinal cord transverse sections at lamina IX illustrating the neuroprotective effect on motoneurons, 4, 8, and 12 weeks following P2 sciatic nerve transection and repair. Note the decreased number of motoneurons ipsilateral to the lesion and the improvement of neuronal survival in the coaptation groups. (a, b, c) Ipsilateral side, 4 weeks after lesion, groups AX, AX+FS, and AX+CFS, respectively. (d) Contralateral side, 4 weeks after lesion. (e, f, g) Ipsilateral side, 8 weeks after lesion, groups AX, AX+FS, and AX+CFS, respectively. (h) Contralateral side, 8 weeks after lesion. (i, j, k) Ipsilateral side, 12 weeks after lesion, groups AX, AX+FS, and AX+CFS, respectively. (l) Contralateral side, 12 weeks after lesion. Scale bar = 50
Quantitative measurements of synaptophysin immunoreactivity in the sciatic motor nuclei after axotomy (AX) and after axotomy followed by coaptation (AX+FS; AX+CFS) were carried out 4, 8, and 12 weeks after injury. Axotomy group showed significant reduction of immunoreactivity ipsilateral to the lesion side. On the contrary, synaptic covering was preserved following coaptation in all survival times analyzed (Figure
Immunohistochemical analysis of the spinal cord ventral horn stained with antisynaptophysin, 4, 8, and 12 weeks, following P2 sciatic nerve transection and repair. A significant preservation of synaptophysin immunoreactivity is observed in both coaptation groups in all time analyzed. (a, b, c) Ipsilateral side, 4 weeks after lesion, groups AX, AX+FS, and AX+CFS, respectively. (d) Contralateral side, 4 weeks after lesion. (e, f, g) Ipsilateral side, 8 weeks after lesion, groups AX, AX+FS, and AX+CFS, respectively. (h) Contralateral side, 8 weeks after lesion. (i, j, k) Ipsilateral side, 12 weeks after lesion, groups AX, AX+FS, and AX+CFS, respectively. (l) Contralateral side, 12 weeks after lesion. Scale bar = 50
A significant increase in astrocyte reactivity after axotomy was observed four weeks after lesion (Figure
Immunohistochemical analysis of the spinal cord ventral horn stained with glial fibrillary acid protein (GFAP), 4, 8, and 12 weeks, following P2 sciatic nerve transection and repair. A decrease in astrogliosis is observed in both coaptation groups, 4 weeks after lesion. (a, b, c) Ipsilateral side, 4 weeks after lesion, groups AX, AX+FS, and AX+CFS, respectively. (d) Contralateral side, 4 weeks after lesion. (e, f, g) Ipsilateral side, 8 weeks after lesion, groups AX, AX+FS, and AX+CFS, respectively. (h) Contralateral side, 8 weeks after lesion. (i, j, k) Ipsilateral side, 12 weeks after lesion, groups AX, AX+FS, and AX+CFS, respectively. (l) Contralateral side, 12 weeks after lesion. Scale bar = 50
Immunohistochemical analysis of the spinal cord ventral horn stained with ionized calcium binding adaptor protein (Iba1), 4, 8, and 12 weeks, following P2 sciatic nerve transection and repair. A decrease in microglial reaction is observed in both coaptation groups, 4 weeks after lesion. (a, b, c) Ipsilateral side, 4 weeks after lesion, groups AX, AX+FS, and AX+CFS, respectively. (d) Contralateral side, 4 weeks after lesion. (e, f, g) Ipsilateral side, 8 weeks after lesion, groups AX, AX+FS, and AX+CFS, respectively. (h) Contralateral side, 8 weeks after lesion. (i, j, k) Ipsilateral side, 12 weeks after lesion, groups AX, AX+FS, and AX+CFS, respectively. (l) Contralateral side, 12 weeks after lesion. Scale bar = 50
Although absence of spontaneous regeneration following axotomy alone was observed, acute coaptation (AX+FS, AX+CFS) resulted in axonal growth with sensorimotor recovery. Area measurements showed no significant difference between AX+FS and AX+CFS groups, although such nerves presented reduced dimensions as compared to the contralateral (nonlesioned) samples (Table
Regenerated sciatic nerve mean area (
Survival time (weeks after lesion) | AX+FS | AX+CFS | Contralateral |
---|---|---|---|
4 | 65,275 ± 12.66 | 43,281 ± 3.07 | 245,655 ± 20.12 |
8 | 68,791 ± 6.93 | 79,175 ± 6.95 | 419,924 ± 54.99 |
12 | 69,526 ± 6.43 | 92,425 ± 13.06 | 420,979 ± 20.77 |
Additionally, the estimation of total number of axons revealed no significant differences between coaptation groups (Figure
Representative micrographs of estimated number of regenerated myelinated axons of the sciatic nerve, 4, 8, and 12 weeks, following P2 sciatic nerve transection and repair. Note smaller fibers and counting in both coaptation groups compared with the contralateral nerve. Axotomy only group did not show any regenerated fiber. (a, b) Ipsilateral side, 4 weeks after lesion, groups AX+FS and AX+CFS, respectively. (c) Contralateral side, 4 weeks after lesion. (d, e) Ipsilateral side, 8 weeks after lesion, groups AX+FS and AX+CFS, respectively. (f) Contralateral side, 8 weeks after lesion. (g, h) Ipsilateral side, 12 weeks after lesion, groups AX+FS and AX+CFS, respectively. (i) Contralateral side, 12 weeks after lesion. Scale bar = 10
Morphometry revealed close to normal fiber distribution frequency following fibrin sealant from CEVAP coaptation. Of note, commercial fibrin adhesive displayed the worst values for all parameters analyzed at four weeks after lesion, indicating slower axonal regeneration progress (Figures
Frequency distribution of fiber diameter of regenerated fibers, 4, 8, and 12 weeks, following P2 sciatic nerve transection and repair. (a, b) Ipsilateral side, 4 weeks after lesion, groups AX+FS and AX+CFS, respectively. (c) Contralateral side, 4 weeks after lesion. (d, e) Ipsilateral side, 8 weeks after lesion, groups AX+FS and AX+CFS, respectively. (f) Contralateral side, 8 weeks after lesion. (g, h) Ipsilateral side, 12 weeks after lesion, groups AX+FS and AX+CFS, respectively. (i) Contralateral side, 12 weeks after lesion. Note smaller diameter fibers in the group AX+CFS, 4 weeks after lesion. Red boxes highlight frequency intervals with greater differences among groups. AX: axotomy; AX+FS: axotomy followed by coaptation with fibrin sealant derived from snake venom; AX+CFS: axotomy followed by coaptation with commercial fibrin sealant.
Frequency distribution of axon diameter of regenerated fibers, 4, 8, and 12 weeks, following P2 sciatic nerve transection and repair. (a, b) Ipsilateral side, 4 weeks after lesion, groups AX+FS and AX+CFS, respectively. (c) Contralateral side, 4 weeks after lesion. (d, e) Ipsilateral side, 8 weeks after lesion, groups AX+FS and AX+CFS, respectively. (f) Contralateral side, 8 weeks after lesion. (g, h) Ipsilateral side, 12 weeks after lesion, groups AX+FS and AX+CFS, respectively. (i) Contralateral side, 12 weeks after lesion. Note smaller axon diameters in the group AX+CFS, 4 weeks after lesion. Red boxes highlight frequency intervals with greater differences among groups. (AX) axotomy; (AX+FS) axotomy followed by coaptation with fibrin sealant derived from snake venom; (AX+CFS) axotomy followed by coaptation with commercial fibrin sealant.
Frequency distribution of myelin thickness of regenerated fibers, 4, 8, and 12 weeks, following P2 sciatic nerve transection and repair. (a, b) Ipsilateral side, 4 weeks after lesion, groups AX+FS and AX+CFS, respectively. (c) Contralateral side, 4 weeks after lesion. (d, e) Ipsilateral side, 8 weeks after lesion, groups AX+FS and AX+CFS, respectively. (f) Contralateral side, 8 weeks after lesion. (g, h) Ipsilateral side, 12 weeks after lesion, groups AX+FS and AX+CFS, respectively. (i) Contralateral side, 12 weeks after lesion. Note decreased myelin thickness in the group AX+CFS, 4 weeks after lesion. Red boxes highlight frequency intervals with greater differences among groups. AX: axotomy; AX+FS: axotomy followed by coaptation with fibrin sealant derived from snake venom; AX+CFS: axotomy followed by coaptation with commercial fibrin sealant.
Statistical differences between groups were only observed at four weeks after sciatic nerve coaptation. In this sense, AX+FS displayed better recovery as compared to AX+CFS. Such statistical evaluation is presented in detail as supplementary material: Figure S4, fiber diameter; Figure S5, axon diameter; and Figure S6, myelin thickness. No statistical differences were depicted regarding the “
Frequency distribution of
All analyzed motoneurons showed at least one cholinergic presynaptic terminal (type C) in apposition to the plasmatic membrane surface. Lesioned neurons, affected by axotomy, showed changes compatible with chromatolysis, such as displacement of the nucleus to the periphery and decrease in cytoplasm electron density. Glial projections were observed, in the AX group, intermingling the space between part of the presynaptic terminals and the postsynaptic membrane (Figure
Representative ultrastructure micrographs showing synapses opposed to
Four weeks after lesion, nontreated neurotmesis (AX group), presented a 41.22% covering reduction of type S inputs when compared with contralateral side. Nerve coaptation resulted in excitatory input preservation, with no statistical differences between commercial and CEVAP sealant (Figures
The gaps between clusters of terminals in apposition to the postsynaptic membrane were measured. Axotomy alone led to greater distance between terminals that contrasted with coaptation repair, where clusters of terminals were close to each other (Figures
The recovery of motor function was studied by the walking track test, using the CatWalk System (Noldus Inc.). Statistical analysis from peroneal functional index showed a significantly greater peroneal functional index (PFI) following coaptation (AX+FS, AX+CFS), regardless the nature of the fibrin sealant (Figure
Motor function recovery up to 12 weeks after lesion by the analysis of the peroneal nerve functional index. Observe the significantly better performance of coaptation groups (AX+FS and AX+CFS) when compared with axotomy without repair group (AX). AX: axotomy; AX+FS: axotomy followed by coaptation with fibrin sealant derived from snake venom; AX+CFS: axotomy followed by coaptation with commercial fibrin sealant; control: unlesioned group.
The nociceptive recovery was analyzed by stimulation of the plantar surface of the paw with electronic von Frey. Contralateral paw showed withdraw reflex around 30 g. Axotomy alone (AX) rats did not show withdraw behavior at the cut-off weight, indicating lack of sensory perception (70 g, Figure
von Frey measurements (
It is well known that neonatal axotomy results in devastating neuronal loss in the spinal cord and dorsal root ganglia (DRG). This is particularly because of the interruption of neurotrophic factors production and retrograde flow, mostly at the target organs. In fact, administration of neurotrophic factors, such as brain derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF) rescue motoneurons from degeneration [
The present work shows that end-to-end nerve coaptation can be performed in a reproducible fashion by using fibrin sealant as a connecting structure between proximal and distal stump. It is important to highlight that in humans nerve size is larger, allowing direct suturing. In this scenario, the use of fibrin glue may enhance repair stability and function as a scaffold for cell migration.
In the present study, we compared two different fibrin adhesives, one commercial brand and another recently developed by CEVAP. The latest is based on nonhuman components, avoiding transmission of infectious blood-borne diseases. The results have shown that both sealants (commercial and CEVAP) are excellent for the coaptation process, although CEVAP’s fibrin sealant has proven to be easier to handle at the moment of surgery. Surgeries involving neonatal subjects ought to be particularly fast, and clotting speed is a crucial parameter. Due to the fact that CEVAP derived adhesive uses bubaline fibrinogen, the concentration of such protein in the cryoprecipitate is specially higher than the human counterpart [
Neuronal death is irreparable and results in sensibility and motor loss. It has been shown that this scenario can be partially reversed by local or systemic administration of trophic substances, such as neurotrophins, antioxidants, cannabinoids, and gangliosides [
Importantly, restauration of neonatal lesion, which mimics obstetric brachial plexus injury, led to preservation of synapses, as seen by synaptophysin immunostaining. Reduction of astrogliosis and microglial reaction also contribute to synapse stability and functionality, correlating with substantial motor and sensory recovery in AX+FS and AX+CFS groups up to twelve weeks after surgery. In this regard, improved peroneal index following coaptation was observed with either fibrin sealant treatment, when compared with axotomy alone. Such improvement indicates reestablishment of muscle innervation as well as suprasegmental control [
After an injury, retraction of presynaptic boutons that appose spinal motoneurons takes place during the first week after lesion. This event reduces synaptic transmission [
Synapse dynamics also depend on astrocytes and microglia that act in favor of maintenance of homeostasis that is necessary to transmission and synaptic plasticity. One well-known retrograde effect of peripheral axotomy is the development of central astrogliosis and microglial reaction, directly interfering on synapse stability [
Overall, the present data suggest that acute repair of neonatal peripheral nerve with both fibrin sealant analyzed, namely, a commercial brand and a nonhuman derived adhesive produced by CEVAP/Brazil, promotes neuroprotection and regeneration of motor and sensory axons. Also, the present study demonstrates that neonatal end-to-end nerve coaptation is feasible and may in turn be of use for repairing obstetric brachial plexus injuries.
The authors declare that there is no conflict of interests regarding the publication of this paper.
The authors acknowledge São Paulo Research Foundation (FAPESP) for providing financial support (2014/06892-3, 2012/19646-6, 2012/08101-8, 2011/23236-4, and 2009/53846-9), the National Council for Scientific and Technological Development (CNPq, Proc. no. 563582/2010-3), and the Coordination for the Improvement of Higher Education Personnel (CAPES, AUXPE Toxinologia1219/2011, Proc. no. 23038.000823/2011-21 and AUXPE Proc. no. 23038.005536/2012-31). Natalia Perussi Biscola received a scholarship from FAPESP (2011/23377-7). Alexandre Leite Rodrigues de Oliveira (300552/2013-9) and Rui Seabra Ferreira Junior (310207/2011-8) received a fellowship from CNPq (Brazil). Special thanks are also extended to the Center for the Study of Venoms and Venomous Animals (CEVAP) of UNESP for enabling the publication of this paper (CAPES, Grant no. 23038.006285/2011–21, AUXPE-Toxinologia, 1219/2011).