Comparison of three different strategies to treat sciatic nerve regeneration: an experimental study

Purpose: To compare the effect of vein conduit filled with adipose tissue stem cells (ASC) on peripheral nerve injury regeneration. Methods: We analyzed 30 male Wistar rats surgically submitted to a 5-mm gap on the sciatic nerve. Then, the animals were divided into three groups: nerve autografting (AG, n=10), autogenous inverted glycerol-conserved vein (VG, n=10), and autogenous inverted glycerol-conserved vein + ASC (VASCG, n=10). The study endpoints were neuromotor functional analysis, gastrocnemius muscle weight, and sciatic nerve graft histomorphometry analysis. In the histologic analysis, we added a control group (naïve nerve). Results: Regarding functional analysis (Walking tract- score), the findings at week 3 showed a difference between the AG and the VG (-96.6 vs. -59.6, p=0.01, respectively) and between the VG and the inverted vein + VASCG (-59.9 vs. -88.92, p=0.02). At week 12, this study showed a difference between the AG and the VG (-64.8 vs. -47.3, p=0.004, respectively), and also a difference between the VG and the VASCG (-47.3 vs. -57.4, p=0.02, respectively). There was no difference in the histomorphometry analysis (nerve diameter, Schwann cells counting). The gastrocnemius muscles on the intervention side were more atrophic when compared to the gastrocnemius muscles on the control side. Conclusions: Our results suggested better functional recovery in the inverted vein group when compared to control group, and inverted vein + ASC group.

Stem cells in end-to-side neurorrhaphy: experimental study in rats

Purpose: To evaluate the influence of mesenchymal stem cells from adipose tissue in the end-to-side neurorrhaphy, focusing in the nerve regeneration and the muscle reinnervation in acute trauma. Methods: 140 animals were randomly divided in seven groups: control, denervated, end-to-side neurorrhaphy between distal stump of common peroneal nerve and tibial nerve (ESN), ESN wrapped in fascia, ESN wrapped in fascia and platelet gel, ESN wrapped in platelet gel, ESN wrapped in fascia and platelet gel within stem cells (without culture) removed from the adipose tissue. Mass measurements of the animal and of cranial tibial muscles, electromyography, walking track analysis tests and histological examinations of the nerves and muscles after 180 days was performed. Results: In the groups where the ESN was performed, the results were always better when compared to the denervated group, showing reinnervation in all ESN groups. The most sensitive methods were walking track and histological analysis. Only the group with stem cells showed values similar to the control group, as well as the functional indices of peroneal nerve and the number of nerve fibers in the peroneal nerve. Conclusions: Stem cells were effective in ESN according with the functional index of the peroneal nerve, evaluated by walking track analysis and the number of nerve fibers in the peroneal nerve.(AU)

Comparative study between standard and inside-out vein graft techniques on sciatic nerve repair of rats: muscular and functional analysis

Purpose: To compare the functional result of standart vein grafts and inside-out vein graft technique on sciatic nerve repair. Methods: We used 24 male Wistar rats divided into 4 groups: control group (CG), standard vein graft group (SVG), Inside-out vein graft group (IOVG) and denervated Group (DG). SVG, IOVG and DG underwent total section of the sciatic nerve, SVG and IOVG however underwent nerve repair surgery using a graft with normal jugular vein and inside-out jugular vein, respectively. Histological analysis of the soleus and Extensor Digitorum Longus (EDL), and Sciatic Functional Index were used to compare the results after 6 weeks. Results: Both grafts acted favorably in muscle recovery and improved functionality; They were similar in all parameters, however, in more points SVG achieved similar to the CG, in the other hand IOVG more times was similar to DG. Fact that makes the graft with normal vein the most viable option between the two options. Conclusion: Both types of grafts acted beneficially wherein the graft normal vein has proved to be the best option.(AU)

Inside-out and standard vein grafts associated with platelet-rich plasma (PRP) in sciatic nerve repair. A histomorphometric study

Purpose: To evaluated the tubulization technique with standard and inside-out vein, filled or not with platelet-rich plasma (PRP), in sciatic nerve repair. Methods: Seventy male Wistar rats were randomly divided into five groups: IOVNF (Inside-Out Vein with No Filling); IOVPRP (Inside-Out Vein filled with PRP); SVNF (Standard Vein with No Filling); SVPRP (Standard Vein filled with PRP); Sham (Control). The left external jugular vein was used as graft in a 10 mm nervous gap. Results: In the morphological analysis of all groups, myelinated nerve fibers with evident myelin sheath, neoformation of the epineurium and perineurium, organization of intraneural fascicles and blood vessels were observed. In the morphometry of the distal stump fibers, SVPRP group had the highest means regarding fiber diameter (3.63±0.42 m), axon diameter (2.37±0.31 m) and myelin sheath area (11.70±0.84 m2). IOVPRP group had the highest means regarding axon area (4.39±1.16 m2) and myelin sheath thickness (0.80±0.19 m). As for values of the fiber area, IOVNF group shows highest means (15.54±0.67 m2), but are still lower than the values of the Sham group. Conclusion: The graft filled with platelet-rich plasma, with use standard (SVPRP) or inside-out vein (IOVPRP), promoted the improvement in axonal regeneration on sciatic nerve injury.(AU)

End-to-side versus end-to-end neurorrhaphy at the peroneal nerve in rats

Purpose: To evaluate three different kinds of neurorrhaphy of the peroneal nerve. Methods: Eigthy rats were divided into 5 groups. Control: nerve had no intervention. End-to-end (EE): nerve was cut and elongated with a nerve graft with two end-to-end neurorrhaphies. End-to-side (ES): nerve was cut and sutured to the graft with at the lateral side of the nerve. Side-to-end (SE): the nerve was cut and sutured to the graft with end-to-end neurorrhaphy. Denervated: nerve was cut and both endings were buried into the muscle. The evaluation was done by walking track analysis, electrophysiology, body mass, cranial tibial muscle mass, nerve and muscle fibers morphometry. Results: The EE, ES and SE have the same potential of reinnervation. Conclusion: There is no functional or histological difference between these different types of neurorrhaphy.(AU)

Combined polyglycolic acid tube and autografting versus autografting or polyglycolic acid tube alone. A comparative study of peripheral nerve regeneration in rats

PURPOSE: To compare sciatic nerve regeneration in rats using three different techniques of repair. METHODS: Fifteen isogonics rats were divided into three groups according to the method used to repair a 5-mm long defect created in the sciatic nerve: autogenous graft (Group A), polyglycolic acid tube (PGAt) (Group B), and of the association of PGAt with the graft (Group C). Histological analysis, regenerated myelinated axon number count and functional analysis were used to compare after six weeks. RESULTS: There was no difference in fiber diameter and degree of myelinization presented by Groups A, B and C. Group B presented the lowest number of regenerated axons. The groups did not display any significant functional difference after walking track analysis (p<0.05). CONCLUSION: No differences between the three groups in terms of functional recovery, although there were histological differences among them.(AU)

INVESTIGAÇÃO IN VITRO E IN VIVO DO EFEITO REGENERATIVO NA MEDULA ESPINAL NA PRESENÇA DE FRAGMENTOS DO NERVO ISQUIÁTICO COM ADIÇÃO DOS ÓLEOS ESSENCIAIS DE Hyptis suaveolens (L.) Poit E Croton blanchetianus Baill

A medula espinal (ME) é afetada em casos de traumas diretos ou indiretos e alguns estudos apontam que os nervos periféricos induzem um ambiente propício para regeneração. Além disso, várias plantas medicinais possuem metabólitos secundários com atividades no Sistema Nervoso Central. Este estudo objetivou analisar o efeito regenerativo na ME in vitro e in vivo na presença de fragmentos do nervo isquiático (FGNI) com adição dos óleos essenciais (OES) de Hyptis suaveolens (HS) e Croton blanchetianus (CB). As plantas de HS e CB foram submetidas ao processo de hidrodestilação e a análise da composição química foi feita por cromatografia gasosa e espectrometria de massas. Para a avaliação da plasticidade na ME in vitro, células foram coletadas de ratos neonatos Wistar e cultivadas em 6 grupos: um controle (D-10) e os demais grupos com diferentes combinações de meio condicionado do nervo isquiático (MCNI) e os OES de HS e CB. A morfometria celular foi avaliada após 96 horas por microscopia de contraste de fases, sendo em seguida realizada a imunocitoquímica para GFAP, GAP-43, NeuN e a plasticidade analisada por Microscopia Eletronica de Varredura. Os grupos foram comparados estatisticamente através do teste de Tukey e Bonferroni (p<0,05). Assim, foi possível avaliar a aderência, o efeito plástico e trófico das células nos grupos cultivados em D-10 acrescidos dos OES, com alterações morfológicas mais visíveis quando comparados com os grupos controle. O OE de CB, especialmente, promoveu a manutenção da expansão das células, indicando sua ação na plasticidade das células da ME. Quanto à análise regenerativa na ME in vivo, foram utilizados 40 ratos Wistar e de 4 deles foi removido o nervo isquiático (NI). Os 36 animais restantes foram submetidos a uma transecção medular com 4 mm de espessura ao nível torácico baixo e divididos em 6 grupos (n=6): no grupo 1 foi inoculado 10l de solução salina (5%) (SS); no grupo 2 FGNI mais 10l de SS (SS + NI); no grupo 3 FGNI mais o OE de HS (HS + NI); no grupo 4 foi inoculado apenas o OE de HS; no grupo 5 FGNI mais o OE de CB (CB + NI) e no grupo 6 foi inoculado apenas o OE de CB. O desempenho dos membros posteriores foi avaliado por 12 semanas usando pontuação do comportamento motor (BBB) e o déficit funcional ligado ao comportamento da pontuação combinada (CBS) e os dados dos testes comportamentais dos grupos tratados foram comparados estatisticamente. Ocorreu recuperação dos movimentos dos membros posteriores dos animais pertencentes aos grupos com o FGNI e com os OES quando comparados ao grupo SS e SS + NI. Entretanto, nos grupos com o OE de CB ocorreram resultados mais significativos e superioridade frente aos grupos com o OE de HS. Portanto, a combinação de OES e nervos periféricos como o NI, in vitro e in vivo, proporcionou a plasticidade morfológica e a melhora da recuperação funcional da ME.

: The spinal cord (SC) is affected in cases of direct or indirect trauma and some studies indicate that the peripheral nerves induce an environment conducive to regeneration. In addition, several medicinal plants have secondary metabolites with activities in the central nervous system. This study aimed to analyze the regenerative effect on SC in vitro and in vivo in the presence of sciatic nerve fragments (SNFG) with the addition of essential oils (EOS) from Hyptis suaveolens (HS) and Croton blanchetianus (CB). The HS and CB plants were submitted to the hydrodistillation process and the analysis of the chemical composition of the EOS was perfomed by gas chromatography and mass spectrometry. For the evaluation of cellular plasticity on SC in vitro, the SC cells were collected from newborn Wistar rats, and then cultured in six groups: one control (D-10) and the others with different combinations of sciatic nerve conditioned medium (SNCM) and HS and CB EOS. Cell morphometry was evaluated after 96 hours by phase contrast microscopy, and then the immunocytochemistry was performed for GFAP, GAP-43, NeuN and plasticity was performed by scanning electron microscopy. The groups were statistically compared using the Tukey and Bonferroni tests (p <0.05). Thus, it was possible to evaluate the adherence, the plastic and trophic effect in groups cultured in D-10 plus EOS, with more visible morphological changes when compared to control groups. CB EO, especially, promoted the maintenance of cell expansion, indicating its action on the plasticity of SC cells. Regarding the regenerative analysis on SC in vivo, forty Wistar rats were used; sciatic nerve was removed from four animals, and thirty-six animals were submitted to a 4 mm thick medullary transection at the lower thoracic level; the animals were later separated in 6 groups (n=6): in group 1, 10l of saline solution (5%)(SS); in group 2, FGNS plus 10l of SS (SS + NS); in group 3, FGNS plus HS essential oil (EO) (HS + NS); in group 4, only HS EO; in group 5, FGNS plus CB EO (CB + NS) and in group 6, only CB EO (CB + NS) were inoculated. Posterior limb performance was assessed weekly for 12 weeks, using motor behavior score (BBB) and functional deficit associated with combined score behavior (CBS). Data from the behavioral tests of the treated groups were compared statistically. The different types of treatments allowed the recovery of the hind limbs movements of the animals of the FGNI and OES groups, and they showed a better recovery when compared to the SS and SS + NS groups. However, the groups with the EO of CB showed more significant results and superiority compared to the groups with the EO of HS. Therefore, the combination of OES and peripheral nerves such as NI, in vitro and in vivo, provided morphological plasticity and an improvement in the functional recovery of SC.

CONDUTO DE ORIENTAÇÃO NERVOSA FUNCIONALIZADO COM CÉLULAS ESTROMAIS MESENQUIMAIS MULTIPOTENTES PROMOVE A REGENERAÇÃO DO NERVO ISQUIÁTICO EM RATOS

As lesões traumáticas do sistema nervoso periférico (SNP) são altamente debilitantes, levando a déficits sensório-motores a longo prazo. A regeneração nervosa depende de um microambiente permissivo para o crescimento axonal, associado a presença de moléculas solúveis bioativas. Neste sentido, o uso das células estromais mesenquimais multipotentes derivadas do tecido adiposo (AdMSC) em combinação com condutos de orientação nervosa (NGC) poderiam acarretar estratégias promissoras para a regeneração nervosa. Ratos Wistar foram submetidos a lesão crítica do nervo isquiático (12 mm de gap) e divididos em grupos experimentais: Sham (abordagem do nervo isquiático sem alterações), GA (técnica de autoenxerto), GPCL (tubulação com NGC vazio), GPCL+MSCc (tubulação com NGC + 106 AdMSC de cão embebidas em biopolímero de fibrina) e GPCL+MSCr (tubulação com NGC + 106 AdMSC de rato embebidas em biopolímero de fibrina). Os NGC de policaprolactona (PCL) foram fabricados por impressão 3D. As AdMSC de cão foram caracterizadas in vitro e foi avaliado o potencial neuroregenerativo após a estimulação com INF-y (BDNF, GDNF, HGF e IL-10). Foram avaliados o índice de funcionalidade do nervo isquiático e tibial durante 12 semanas in vivo. A análise da marcha e a eletroneuromiografia foram avaliadas nas semanas 8 e 12. Foi realizada a morfometria post-mortem nas semanas 8 e 12 após o reparo nervoso. Trinta dias após a lesão nos grupos GPCL+MSCc e Sham, foram analisadas a expressão de fatores neurotróficos (BDNF, GDNF e o receptor p75NTR) e a reatividade das células de Schwann (S-100 e neurofilamento) pela imunofluorescencia, e a expressão gênica na medula espinhal (BDNF, GDNF, HGF, IL-6 e IL-10). A estimulação com INF-y aumentou a expressão gênica de BDNF, GDNF, HGF e IL-10 nas AdMSC de cão. Os grupos GPCL+MSCc e GPCL+MSCr apresentaram recuperação motora e eletrofisiológica quando comparados ao grupo GPCL em 8 e 12 semanas. Porém, os valores não foram superiores ao autoenxerto. Os achados foram relacionados a presença de fibras mielinizadas, maior expressão de BDNF, GDNF e P75NTR no nervo isquiático e a up-regulation de BDNF, GDNF e HGF na medula espinhal. Foi observada tendência a maior reatividade das células de Schwann e ramificação axonal no nervo isquiático. A combinação de NGC impressos em 3D e funcionalizados com AdMSC de cão e de rato mostraram efeitos neuroprotetores. A associação de AdMSC e biopolímero de fibrina suportaram o microambiente trófico e estimularam o estado pró-regenerativo na lesão crítica do nervo isquiático em ratos.

Peripheral nervous injuries (PNS) are highly debilitating, leading to long-term motor and sensory deficits. Nerve regeneration depends of the permissive microenvironment for axonal growth associated to bioactive molecules. In this sense, the use of multipotent mesenchymal stromal cells adipose tissue derived (AdMSC) in combination with nerve guidance conduits (NGC) could be promising strategies for nerve regeneration. Wistar rats were submitted to critical sciatic nerve injury (12 mm gap) and divided into experimental groups: Sham, GA (autograft), GPCL (empty NGC), GPCL + MSCc (NGC + 106 plus canine AdMSC embedded in fibrin biopolymer), GPCL + MSCr (NGC + 106 plus rat AdMSC embedded in fibrin biopolymer). Polycaprolactone (PCL) NGC was manufactured by 3D printing. In vitro, regenerative potential was evaluated after INF- stimulation (BDNF, GDNF, HGF and IL-10). In vivo, were measure the sciatic ant tibial functional index for 12 weeks. Gait analysis and electroneuromyography were performed at 8 and 12 weeks. Post-mortem, morphometric analysis was made at 8 and 12 weeks after nerve repair. Thirty days after lesion in the GPLC+MSCc and Sham, were evaluated the expression of neurotrophins (BDNF, GDNF and p75NTR receptor), Schwann cells reactivity by immunofluorescence (S-100 and neurofilament) and gene expression in the spinal cord (BDNF, GDNF, HGF, IL-6 and IL-10). The stimulation with INF- increased gene expression on canine AdMSC in vitro for BDNF, GDNF, HGF and IL-10. The groups GPCL+MSCc and GPCL+MSCr showed functional motor and electrophysiological motor recovery when compared with the GPCL group at 8 and 12 weeks. However, the values were not superior to GA group. These findings were related to presence of myelinated fibers, increased of neurotrophins expression BDNF, GDNF and p75NTR on sciatic nerve and up-regulation of the BDNF, GDNF and HGF in the spinal cord. A trend towards greater reactivity of Schwann cells and axonal branching in the sciatic nerve were observed. The combination of NGC 3D-printing and functionalized with canine and rat AdMSC showed neuroprotective effects. The association of canine or rat AdMSC and fibrin biopolymer support trophic microenvironment and stimulate a pro-regenerative state in severe sciatic nerve injury in rats.

Evaluation of nerve regeneration in diabetic rats

PURPOSE: To compare sciatic nerve regeneration between non-diabetic (control) and streptozotocin-induced diabetic Wistar rats. METHODS:Four subgroups were evaluated. CN: Non-diabetic rats submitted to neurorrhaphy (n=9); DN: Diabetic rats submitted to neurorrhaphy (n=9); CG: Non-diabetic rats submitted to nerve grafting (n=10); DG: Diabetic rats submitted to nerve grafting (n=9). The nerve regeneration was evaluated by walking track analysis (sciatic functional index), electrophysiological test, histomorphometric analysis and triceps surae muscle weight. RESULTS:At 60 days post-surgery, functional recovery of DN was similar to that of the non-diabetic rats (CN, CG), but DG didn't achieve the same. Evoked potential amplitudes showed no statistically significant differences among subgroups. Triceps surae muscle was heavier in CN. No statistically significant differences were observed between the control and diabetes subgroups with respect to histomorphometric analysis. CONCLUSION: After 60 days, DN had a functionally similar recovery to that of the control animals, whereas nerve grafting in diabetic rats didn't allow the same. The muscle atrophy was lower in CN. In the rest of evaluations, as electrophysiological and histomorphometric, diabetic rats were not different from control ones.(AU)

Peroneal nerve reconstruction by using glycerol-preserved veins: histological and functional assessment in rats

PURPOSE: To compare the degree of neural regeneration in rats upon interposition of autologous nerve graft, autogenous vein, glycerol-preserved autogenous vein, and glycerol-preserved allogeneic vein using qualitative and quantitative histological analyses as well as functional assessments. METHODS: Peroneal nerves were reconstructed differently in four groups of animals. Functional assessments were performed pre- and postoperatively for a period of six weeks. After six weeks, the animals were sacrificed and histological evaluations were performed. RESULTS: Histological patterns of autogenous veins without preservation showed pronounced neoangiogenesis and extensive axonal rarefaction, as confirmed by axonal counting and functional assessments. Glycerol-preserved veins had results similar to the control. CONCLUSIONS: Glycerol-preserved autogenous or allogeneic veins showed similar results to autograft results. The autogenous vein (without preservation in glycerol) presented histological and functional outcomes statistically lower than other groups.(AU)