Neuroprotection and immunomodulation following intraspinal axotomy of motoneurons by treatment with adult mesenchymal stem cells.

BACKGROUND: Treatment of spinal cord injury is dependent on neuronal survival, appropriate synaptic circuit preservation, and inflammatory environment management. In this sense, mesenchymal stem cell (MSC) therapy is a promising tool that can reduce glial reaction and provide trophic factors to lesioned neurons. METHODS: Lewis adult female rats were submitted to a unilateral ventral funiculus cut at the spinal levels L4, L5, and L6. The animals were divided into the following groups: IA (intramedullary axotomy), IA + DMEM (Dulbecco's modified Eagle's medium), IA + FS (fibrin sealant), IA + MSC (106 cells), and IA + FS + MSC (106 cells). Seven days after injury, qPCR (n = 5) was performed to assess gene expression of VEGF, BDNF, iNOS2, arginase-1, TNF-α, IL-1ß, IL-6, IL-10, IL-4, IL-13, and TGF-ß. The cellular infiltrate at the lesion site was analyzed by hematoxylin-eosin (HE) staining and immunohistochemistry (IH) for Iba1 (microglia and macrophage marker) and arginase-1. Fourteen days after injury, spinal alpha motor neurons (MNs), evidenced by Nissl staining (n = 5), were counted. For the analysis of astrogliosis in spinal lamina IX and synaptic detachment around lesioned motor neurons (GAP-43-positive cells), anti-GFAP and anti-synaptophysin immunohistochemistry (n = 5) was performed, respectively. Twenty-eight days after IA, the gait of the animals was evaluated by the walking track test (CatWalk; n = 7). RESULTS: The site of injury displayed strong monocyte infiltration, containing arginase-1-expressing macrophages. The FS-treated group showed upregulation of iNOS2, arginase-1, proinflammatory cytokine (TNF-α and IL-1ß), and antiinflammatory cytokine (IL-10, IL-4, and IL-13) expression. Thus, FS enhanced early macrophage recruitment and proinflammatory cytokine expression, which accelerated inflammation. Rats treated with MSCs displayed high BDNF-positive immunolabeling, suggesting local delivery of this neurotrophin to lesioned motoneurons. This BDNF expression may have contributed to the increased neuronal survival and synapse preservation and decreased astrogliosis observed 14 days after injury. At 28 days after lesion, gait recovery was significantly improved in MSC-treated animals compared to that in the other groups. CONCLUSIONS: Overall, the present data demonstrate that MSC therapy is neuroprotective and, when associated with a FS, shifts the immune response to a proinflammatory profile.

Impairment of toll-like receptors 2 and 4 leads to compensatory mechanisms after sciatic nerve axotomy.

BACKGROUND: Peripheral nerve injury results in retrograde cell body-related changes in the spinal motoneurons that will contribute to the regenerative response of their axons. Successful functional recovery also depends on molecular events mediated by innate immune response during Wallerian degeneration in the nerve microenvironment. A previous study in our lab demonstrated that TLR 2 and 4 develop opposite effects on synaptic stability in the spinal cord after peripheral nerve injury. Therefore, we suggested that the better preservation of spinal cord microenvironment would positively influence distal axonal regrowth. In this context, the present work aimed to investigate the influence of TLR2 and TLR4 on regeneration and functional recovery after peripheral nerve injury. METHODS: Eighty-eight mice were anesthetized and subjected to unilateral sciatic nerve crush (C3H/HeJ, n = 22, C3H/HePas, n = 22; C57Bl6/J, n = 22 and TLR2(-/-), n = 22). After the appropriate survival times (3, 7, 14 days, and 5 weeks), all mice were killed and the sciatic nerves and tibialis cranialis muscles were processed for immunohistochemistry and transmission electron microscopy (TEM). Gait analysis, after sciatic nerve crushing, was performed in another set of mice (minimum of n = 8 per group), by using the walking track test (CatWalk system). RESULTS: TLR4 mutant mice presented greater functional recovery as well as an enhanced p75(NTR) and neurofilament protein expression as compared to the wild-type strain. Moreover, the better functional recovery in mutant mice was correlated to a greater number of nerve terminal sprouts. Knockout mice for TLR2 exhibited 30 % greater number of degenerated axons in the distal stump of the sciatic nerve and a decreased p75(NTR) and neurofilament protein expression compared to the wild type. However, the absence of TLR2 receptor did not influence the overall functional recovery. End-point equivalent functional recovery in transgenic mice may be a result of enhanced axonal diameter found at 2 weeks after lesion. CONCLUSIONS: Altogether, the present results indicate that the lack of TLR2 or the absence of functional TLR4 does affect the nerve regeneration process; however, such changes are minimized through different compensatory mechanisms, resulting in similar motor function recovery, as compared to wild-type mice. These findings contribute to the concept that innate immune-related molecules influence peripheral nerve regeneration by concurrently participating in processes taking place both at the CNS and PNS.

Alpha motoneurone input changes in dystrophic MDX mice after sciatic nerve transection.

BACKGROUND: Duchenne muscular dystrophy (DMD) is a severe form of muscular dystrophy. At present, a lot is known about the muscular degeneration in DMD, but few studies have focused on the effects on the central nervous system. In this sense, retrograde changes in the microenvironment around motor neurones in the spinal cord may contribute to the pathogenesis of the dystrophinopathies. AIMS: The aim of this study was to investigate synaptic alterations and glial reactivity in the microenvironment close to spinal motor neurones in a DMD animal model. METHODS: Six-week-old male MDX mice were subjected to left sciatic nerve transection. The axotomy was performed after the muscular degeneration/regeneration cycles previously described in such animal models. C57BL/10 mice were used as the control. Seven days after surgery, the animals were sacrificed and the lumbar spinal cords processed for immunohistochemistry using antibodies to the major histocompatibility complex of class I (MHC I), synaptophysin, IBA-1 and glial fibrillary acidic protein (GFAP). RESULTS: MHC I expression increased in both strains after axotomy. Nevertheless, the MDX mice displayed significantly lower MHC I up-regulation. With respect to GFAP expression, the MDX mice showed greater astrogliosis as compared with C57BL/10 mice. The MDX mice displayed a significant decrease in synaptophysin expression. Indeed, the ultrastructural quantitative analysis showed more intense synaptic detachment in MDX mice, indicating a reduction in synaptic activity before and after axotomy. CONCLUSIONS: The reduction in active inputs and increased gliosis in MDX mice may be associated with the muscle degeneration/regeneration cycles that occur postnatally, and could contribute to the seriousness of the disease.

Interferon (IFN) beta treatment induces major histocompatibility complex (MHC) class I expression in the spinal cord and enhances axonal growth and motor function recovery following sciatic nerve crush in mice.

AIMS: Major histocompatibility complex (MHC) class I expression by neurones and glia constitutes an important pathway that regulates synaptic plasticity. The upregulation of MHC class I after treatment with interferon beta (IFN beta) accelerates the response to injury. Therefore the present work studied the regenerative outcome after peripheral nerve lesion and treatment with IFN beta, aiming at increasing MHC class I upregulation in the spinal cord. METHODS: C57BL/6J mice were subjected to unilateral sciatic nerve crush and treatment with IFN beta. The lumbar spinal cords were processed for immunohistochemistry, in situ hybridization, Western blotting and RT-PCR, while the sciatic nerves were submitted for immunohistochemistry, morphometry and counting of regenerated axons. Motor function recovery was monitored using the walking track test. RESULTS: Increased MHC class I expression in the motor nucleus of IFN beta-treated animals was detected. In the peripheral nerve, IFN beta-treated animals showed increased S100, GAP-43 and p75NTR labelling coupled with a significantly greater number of regenerated axons. No significant differences were found in neurofilament or laminin labelling. The morphological findings, indicating improvements in the regenerative process after IFN treatment were in line with the motor behaviour test applied to the animals during the recovery process. CONCLUSIONS: The present data reinforce the role of MHC class I upregulation in the response to injury, and suggest that IFN treatment may be beneficial to motor recovery after axotomy.

Neuroprotective effects of mesenchymal stem cells on spinal motoneurons following ventral root axotomy: synapse stability and axonal regeneration.

Compression of spinal roots is an important medical problem, which may arise from intervertebral disc herniation, tumor growth or as a result of high energy accidents. Differently from avulsion, root crushing maintains the central/peripheral nervous system (CNS/PNS) connection, although the axons are axotomized and motoneurons degenerate. Such neuronal death may decrease and delay motor function recovery. In the present study we have investigated the neuroprotective effects of mesenchymal stem cell (MSC) therapy following such proximal lesions. Motor recovery and synaptic stabilization were analyzed by the use of morphological and functional approaches. For that, crushing the ventral roots at L4, L5 and L6 was unilaterally performed in Lewis rats. Four weeks after injury, an increased motoneuron survival was observed in the MSC-treated group, coupled with a smaller decrease of inputs at the motoneuron surface and nearby neuropil, seen by synaptophysin and synapsin immunolabeling and decreased astrogliosis, seen by GFAP immunolabeling. In this sense, MSC-treated group displayed a significant preservation of GABAergic terminals, indicating a possible neuroprotection to glutamate excitotoxicity. Motor function recovery was acutely improved in MSC-treated group as compared to Dulbeco's modified eagle medium (DMEM)-treated. Overall, we provide evidence that ventral root crushing (VRC), although milder than avulsion, results in significant loss of motoneurons (~51%) that can be reduced by MSC administration within the spinal cord. Such treatment also improves the number of synapses immunoreactive against molecules present in inhibitory inputs. Also, an increased number of regenerated axons was obtained in the MSC-treated group, in comparison to the DMEM-treated control. Overall, MSC therapy acutely improved limb strength and gait coordination, indicating a possible clinical application of such treatment following proximal lesions at the CNS/PNS interface.

Arrabidaea chica extract improves gait recovery and changes collagen content during healing of the Achilles tendon.

INTRODUCTION: Tendon lesions are still a serious clinical problem. The leaves of the Bignoniaceae Arrabidaea chica (Humb. & Bonpl.) B. Verlot. (syn. Bignonia chica (Bonpl.)) have been used in traditional medicine and described in the literature for its healing properties. However, no study has shown the effects of A. chica during tendon healing. The aim of this study was to investigate the healing properties of the A. chica leaves extract on tendons after partial transection. METHODS: A partial transection in the tension region of the Achilles tendon of rats was performed with subsequent posterior topical application of A. chica extract (2.13g/mL in 0.85% saline solution) at the site of the injury. The animals (n=154) were separated into 7 groups: N - rats with tendons without transection; S7, S14 and S21 - rats with tendons treated with topical applications of saline for 7 days and sacrificed on the 7th, 14th and 21st days after surgery, respectively; A7, A14 and A21 - rats with tendons treated with topical applications of the plant extract. The transected regions of the tendons were analyzed through biochemical, morphological and functional analyses. To evaluate the type and concentration of collagen, Western blotting for collagen types I and III was performed, and the hydroxyproline concentration was determined. The participation of metalloproteinases (MMP)-2 and -9 during tendon remodelling was investigated through zymography. Gait recovery was analyzed using the catwalk system. The organization of the extracellular matrix and morphometry were detected in sections stained with haematoxylin-eosin. RESULTS: The application of A. chica extract in the region of tendon injury led to an increase in the amount of hydroxyproline (mg/g tissue) on the 7th (91.5±18.9) and 21st (95.8±11.9) days after the tendon lesion relative to the control groups treated with saline (S7: 75.2±7.2; and S21: 71.9±7.9). There were decreases in collagen types I and III (as determined by densitometry) in the groups treated with the plant extract 7 days after injury (type I: 103.9±15.9; type III: 206.3±8.1) compared to the saline-treated groups (type I: 165.2±31.1; type III: 338.6±48.8). The plant extract stimulated the synthesis of MMP-2 on the 21st day after the lesion and decreased the amount of latent and active isoforms of MMP-9 on the 14th day. Analysis by the catwalk system (max contact intensity) showed that the A. chica extract improved the gait of rats on the 7th day of the healing process when compared to the saline group. CONCLUSIONS: The use of A. chica extract during the healing process of the tendon leads to an increase in collagen content and improved gait recovery. Further studies will be performed to analyze the effect of this plant extract on the organization of the collagen bundles of tendons after lesions and to study its probable anti-inflammatory effect.

Supraorganized collagen enhances Schwann cell reactivity and organization in vitro.

We investigated the reactivity and expression of basal lamina collagen by Schwann cells (SCs) cultivated on a supraorganized bovine-derived collagen substrate. SC cultures were obtained from sciatic nerves of neonatal Sprague-Dawley rats and seeded on 24-well culture plates containing collagen substrate. The homogeneity of the cultures was evaluated with an SC marker antibody (anti-S-100). After 1 week, the cultures were fixed and processed for immunocytochemistry by using antibodies against type IV collagen, S-100 and p75NTR (pan neurotrophin receptor) and for scanning electron microscopy (SEM). Positive labeling with antibodies to the cited molecules was observed, indicating that the collagen substrate stimulates SC alignment and adhesion (collagen IV labeling - organized collagen substrate: 706.33 ± 370.86, non-organized collagen substrate: 744.00 ± 262.09; S-100 labeling - organized collagen: 3809.00 ± 120.28, non-organized collagen: 3026.00 ± 144.63, P < 0.05) and reactivity (p75NTR labeling - organized collagen: 2156.33 ± 561.78, non-organized collagen: 1424.00 ± 405.90, P < 0.05; means ± standard error of the mean in absorbance units). Cell alignment and adhesion to the substrate were confirmed by SEM analysis. The present results indicate that the collagen substrate with an aligned suprastructure, as seen by polarized light microscopy, provides an adequate scaffold for SCs, which in turn may increase the efficiency of the nerve regenerative process after in vivo repair.

Major histocompatibility complex class I expression and glial reaction influence spinal motoneuron synaptic plasticity during the course of experimental autoimmune encephalomyelitis.

Recent studies have shown that major histocompatibility complex class I (MHC I) expression directly influences the stability of nerve terminals. Also, the acute phase of experimental autoimmune encephalomyelitis (EAE) has shown a significant impact on inputs within the spinal cord. Therefore, the present work investigated the synaptic covering of motoneurons during the induction phase of disease and progressive remissions of EAE. EAE was induced in C57BL/6J mice, which were divided into four groups: normal, peak disease, first remission, and second remission. The animals were killed and their lumbar spinal cords processed for in situ hybridization (IH), immunohistochemistry, and transmission electron microscopy (TEM). The results indicated an increase in glial reaction during the peak disease. During this period, the TEM analysis showed a reduction in the synaptic covering of the motoneurons, corresponding to a reduction in synaptophysin immunolabeling and an increase in the MHC I expression. The IH analysis reinforced the immunolabeling results, revealing an increased expression of MHC I mRNA by motoneurons and nonneuronal cells during the peak disease and first remission. The results observed in both remission groups indicated a return of the terminals to make contact with the motoneuron surface. The ratio between excitatory and inhibitory inputs increased, indicating the potential for development of an excitotoxic process. In conclusion, the results presented here indicate that MHC I up-regulation during the course of EAE correlates with the periods of synaptic plasticity induced by the infiltration of autoreactive immune cells and that synaptic plasticity decreases after recurrent peaks of inflammation.

Expression of basal lamina components by Schwann cells cultured on poly(lactic acid) (PLLA) and poly(caprolactone) (PCL) membranes.

The present in vitro study investigated the expression of basal lamina components by Schwann cells (SCs) cultivated on PCL and PLLA membranes prepared by solvent evaporation. Cultures of SCs were obtained from sciatic nerves from neonatal Sprague Dawley rats and seeded on 24 well culture plates containing the polymer membranes. The purity of the cultures was evaluated with a Schwann cell marker antibody (anti-S-100). After one week, the cultures were fixed and processed for immunocytochemistry by using antibodies against type IV collagen, laminin I and II. Positive labeling against the studied molecules was observed, indicating that such biomaterials positively stimulate Schwann cell adhesion and proliferation. Overall, the present results provide evidence that membrane-derived biodegradable polymers, particularly those derived from PLLA, are able to provide adequate substrate and stimulate SCs to produce ECM molecules, what may have in turn positive effects in vivo, influencing the peripheral nerve regeneration process.

The immunomodulator glatiramer acetate influences spinal motoneuron plasticity during the course of multiple sclerosis in an animal model.

The immunomodulador glatiramer acetate (GA) has been shown to significantly reduce the severity of symptoms during the course of multiple sclerosis and in its animal model--experimental autoimmune encephalomyelitis (EAE). Since GA may influence the response of non-neuronal cells in the spinal cord, it is possible that, to some extent, this drug affects the synaptic changes induced during the exacerbation of EAE. In the present study, we investigated whether GA has a positive influence on the loss of inputs to the motoneurons during the course of EAE in rats. Lewis rats were subjected to EAE associated with GA or placebo treatment. The animals were sacrificed after 15 days of treatment and the spinal cords processed for immunohistochemical analysis and transmission electron microscopy. A correlation between the synaptic changes and glial activation was obtained by performing labeling of synaptophysin and glial fibrillary acidic protein using immunohistochemical analysis. Ultrastructural analysis of the terminals apposed to alpha motoneurons was also performed by electron transmission microscopy. Interestingly, although the GA treatment preserved synaptophysin labeling, it did not significantly reduce the glial reaction, indicating that inflammatory activity was still present. Also, ultrastructural analysis showed that GA treatment significantly prevented retraction of both F and S type terminals compared to placebo. The present results indicate that the immunomodulator GA has an influence on the stability of nerve terminals in the spinal cord, which in turn may contribute to its neuroprotective effects during the course of multiple sclerosis.

MHC I upregulation influences astroglial reaction and synaptic plasticity in the spinal cord after sciatic nerve transection.

Recent studies suggested that the MHC class I expression has an important role on the maintenance of synaptic connections and also on neuronal/glial communication. IFN beta is a cytokine that influences the MHC class I expression. Therefore, the present work studied the effects of IFN beta on astrocyte reactivity and synaptic plasticity in the spinal cord. C57BL/6J adult mice were subjected to unilateral sciatic nerve transection after being treated with 10,000 IU of IFN beta for 1 week. Following axotomy, they were kept under treatment for another week. After that, the animals were sacrificed and the lumbar spinal cords were processed for immunohistochemistry and electron microscopy. Placebo and non-treated axotomized groups were used as controls. The results showed an upregulation of GFAP expression in the lesioned spinal cord segments, especially in the IFN treated group. Interestingly, IFN treated animals, showed a grater MHC class I expression coupled with a decrease of synapthophysin immunoreactivity. The ultrastructure of synapses showed a larger pruning of presynaptic terminals in contact with alpha motoneurons, induced by axotomy plus IFN beta treatment. In vitro, primary cultures of astrocytes were treated during 1 week with IFN (non-treated, 100, 500 and 1,000 IU/ml) and processed for immunohistochemistry (GFAP, ezrin and OX-18). They showed a sharp upregulation of GFAP, mostly when subjected to 500 and 1,000 IU. The present results reinforce the role of MHC class I upregulation on the response to injury, both in vivo and in vitro.

Spinal motoneuron synaptic plasticity during the course of an animal model of multiple sclerosis.

During the course of experimental autoimmune encephalomyelitis, a massive loss of motor and sensitive function occurs, which has been classically attributed to the demyelination process. In rats, the clinical signs disappear within 5 days following complete tetraplegia, indicating that demyelination might not be the only cause for the rapid evolution of the disease. The present work investigated the occurrence of experimental autoimmune encephalomyelitis-induced changes of the synaptic covering of spinal motoneurons during exacerbation and after remission. The terminals were typed with transmission electron microscopy as C-, F- and S-type. Immunohistochemical analysis of synaptophysin, glial fibrillary acidic protein and the microglia/macrophage marker F4/80 were also used in order to draw a correlation between the synaptic changes and the glial reaction. The ultrastructural analysis showed that, during exacerbation, there was a strong retraction of both F- and S-type terminals. In this sense, both the covering as well as the length of the remaining terminals suffered great reductions. However, the retracted terminals rapidly returned to apposition, although the mean length remained shorter. A certain level of sprouting may have occurred as, after remission, the number of F-terminals was greater than in the control group. The immunohistochemical analysis showed that the peak of synaptic loss was coincident with an increased macro- and microglial reaction. Our results suggest that the major changes occurring in the spinal cord network during the time course of the disease may contribute significantly to the origin of the clinical signs as well as help to explain their rapid recovery.

Supraorganized collagen enhances Schwann cell reactivity and organization in vitro

We investigated the reactivity and expression of basal lamina collagen by Schwann cells (SCs) cultivated on a supraorganized bovine-derived collagen substrate. SC cultures were obtained from sciatic nerves of neonatal Sprague-Dawley rats and seeded on 24-well culture plates containing collagen substrate. The homogeneity of the cultures was evaluated with an SC marker antibody (anti-S-100). After 1 week, the cultures were fixed and processed for immunocytochemistry by using antibodies against type IV collagen, S-100 and p75NTR (pan neurotrophin receptor) and for scanning electron microscopy (SEM). Positive labeling with antibodies to the cited molecules was observed, indicating that the collagen substrate stimulates SC alignment and adhesion (collagen IV labeling - organized collagen substrate: 706.33 ± 370.86, non-organized collagen substrate: 744.00 ± 262.09; S-100 labeling - organized collagen: 3809.00 ± 120.28, non-organized collagen: 3026.00 ± 144.63, P < 0.05) and reactivity (p75NTR labeling - organized collagen: 2156.33 ± 561.78, non-organized collagen: 1424.00 ± 405.90, P < 0.05; means ± standard error of the mean in absorbance units). Cell alignment and adhesion to the substrate were confirmed by SEM analysis. The present results indicate that the collagen substrate with an aligned suprastructure, as seen by polarized light microscopy, provides an adequate scaffold for SCs, which in turn may increase the efficiency of the nerve regenerative process after in vivo repair.

The immunomodulator glatiramer acetate influences spinal motoneuron plasticity during the course of multiple sclerosis in an animal model

The immunomodulador glatiramer acetate (GA) has been shown to significantly reduce the severity of symptoms during the course of multiple sclerosis and in its animal model - experimental autoimmune encephalomyelitis (EAE). Since GA may influence the response of non-neuronal cells in the spinal cord, it is possible that, to some extent, this drug affects the synaptic changes induced during the exacerbation of EAE. In the present study, we investigated whether GA has a positive influence on the loss of inputs to the motoneurons during the course of EAE in rats. Lewis rats were subjected to EAE associated with GA or placebo treatment. The animals were sacrificed after 15 days of treatment and the spinal cords processed for immunohistochemical analysis and transmission electron microscopy. A correlation between the synaptic changes and glial activation was obtained by performing labeling of synaptophysin and glial fibrillary acidic protein using immunohistochemical analysis. Ultrastructural analysis of the terminals apposed to alpha motoneurons was also performed by electron transmission microscopy. Interestingly, although the GA treatment preserved synaptophysin labeling, it did not significantly reduce the glial reaction, indicating that inflammatory activity was still present. Also, ultrastructural analysis showed that GA treatment significantly prevented retraction of both F and S type terminals compared to placebo. The present results indicate that the immunomodulator GA has an influence on the stability of nerve terminals in the spinal cord, which in turn may contribute to its neuroprotective effects during the course of multiple sclerosis.

Apoptosis of sensory neurons and satellite cells after sciatic nerve transection in C57BL/6J mice

The rate of axonal regeneration, after sciatic nerve lesion in adult C57BL/6J mice, is reduced when compared to other isogenic strains. It was observed that such low regeneration was not due just to a delay, since neuronal death was observed. Two general mechanisms of cell death, apoptosis and necrosis, may be involved. By using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) technique, we demonstrated that a large number of sensory neurons, as well as satellite cells found in the dorsal root ganglia, were intensely labeled, thus indicating that apoptotic mechanisms were involved in the death process. Although almost no labeled neurons or satellite cells were observed one week after transection, a more than ten-fold increase in TUNEL labeling was detected after two weeks. The results obtained with the C57BL/6J strain were compared with those of the A/J strain, which has a much higher peripheral nerve regeneration potential. In A/J mice, almost no labeling of sensory neurons or satellite cells was observed after one or two weeks, indicating the absence of neuronal loss. Our data confirm previous observations that approximately 40 percent of C57BL/6J sensory neurons die after sciatic nerve transection, and indicate that apoptotic events are involved. Also, our observations reinforce the hypothesis that the low rate of axonal regeneration occurring in C57BL/6J mice may be the result of a mismatch in the timing of the neurons need for neurotrophic substances, and production of the latter by non-neuronal cells in the distal stump

Non-neuronal cells are not the limiting factor for the low axonal regeneration in C57BL/6J mice

Peripheral axonal regeneration was investigated in adult male mice of the C57BL/6J (C), BALB/cJ (B) and A/J (A) strains and in their F1 descendants using a predegenerated nerve transplantation model. Four types of transplants were performed: 1) isotransplants between animals of the C, B and A strains; 2) donors of the C strain and recipients of the C x B and C x A breeding; 3) donors of the B strain and recipients of the C x B breeding, and 4) donors of the A strain and recipients of the C x A breeding. Donors had the left sciatic nerve transected and two weeks later a segment of the distal stump was transplanted into the recipient. Four weeks after transplantation the regenerated nerves were used to determine the total number of regenerated myelinated fibers (TMF), diameter of myelinated fibers (FD) and myelin thickness (MT). The highest TMF values were obtained in the groups where C57BL/6J mice were the donors (C to F1 (C x B) = 4658 + OR - 304; C to F1 (C x A) = 3899 + OR - 198). Also, A/J grafts led to a significantly higher TMF (A to F1 (C x A) = 3933 + OR - 565). Additionally, isotransplant experiments showed that when the nerve is previously degenerated, C57BL/6J mice display the largest number of myelinated fibers (C to C = 3136 + OR - 287; B to B = 2759 + OR - 170, and A to A = 2835 + OR - 239). We also observed that when C57BL/6J was the graft donor, FD was the highest and MT did not differ significantly when compared with the other groups. These morphometric results reinforce the idea that Schwann cells and the nerve environment of C57BL/6J provide enough support to the regenerative process. In this respect, the present results support the hypothesis that the non-neuronal cells, mainly Schwann cells, present in the sciatic nerve of C57BL/6J mice are not the main limiting factor responsible for low axonal regeneration