Local neurotoxicity and myotoxicity evaluation of cyclodextrin complexes of bupivacaine and ropivacaine.

BACKGROUND: Bupivacaine (BVC) and ropivacaine (RVC) are local anesthetics widely used in surgical procedures. In previous studies, inclusion complexes of BVC or RVC in hydroxypropyl-ß-cyclodextrin (HP-ß-CD) increased differential nervous blockade, compared to the plain anesthetic solutions. In this study we evaluated the local neural and muscular toxicity of these new formulations containing 0.5% BVC or RVC complexed with HP-ß-CD (BVC(HP-ß-CD) and RVC(HP-ß-CD)). METHODS: Schwann cell viability was assessed by determination of mitochondrial dehydrogenase activity, and histopathological evaluation of the rat sciatic nerve was used to identify local neurotoxic effects (48 hours and 7 days after the treatments). Evaluations of serum creatine kinase levels and the histopathology of rat gastrocnemius muscle (48 hours after treatment) were also performed. RESULTS: Schwann cell toxicity evaluations revealed no significant differences between complexed and plain local anesthetic formulations. However, use of the complexed local anesthetics reduced serum creatine kinase levels 5.5-fold, relative to the plain formulations. The differences were significant at P < 0.05 (BVC) and P < 0.01 (RVC). The histopathological muscle evaluation showed that differences between groups treated with local anesthetics (BVC or RVC) and their respective complexed formulations (BVC(HP-ß-CD) or RVC(HP-ß-CD)) were significant (P < 0.05). CONCLUSIONS: We concluded that the new formulations presented a lower myotoxicity and a similar cytotoxic effect when compared to plain local anesthetic solutions.

Interferon beta and glatiramer acetate induce proliferation of Schwann cells in vitro.

Techniques as well as substances capable of stimulating cultured Schwann cell (SC) proliferation are needed for future therapeutical applications. In this work, the effects of interferon beta (IFNbeta) and glatiramer acetate (GA) on SC cultures were tested, with interest on the growth curve and potential proliferative effects. Primary cultures were prepared from the sciatic nerves of neonatal rats and seeded onto culture plates. Such cells were then subjected to treatment with different doses of IFN beta (100, 500 and 1000 IU/ml) and of GA (1.2, 2.5 and 5.0 microg/ml) for five consecutive days. S100beta and DAPI double labeling was used in order to confirm the purity of the cultures. Both treatment with IFN and GA resulted in an increased number of cultured SCs. However, only IFN beta induced a statistically significant proliferative outcome. Such results indicate that addition of IFN beta to the culture medium is efficient in order to improve SC proliferation in vitro.

Pharmacological and local toxicity studies of a liposomal formulation for the novel local anaesthetic ropivacaine.

This study reports an investigation of the pharmacological activity, cytotoxicity and local effects of a liposomal formulation of the novel local anaesthetic ropivacaine (RVC) compared with its plain solution. RVC was encapsulated into large unilamellar vesicles (LUVs) composed of egg phosphatidylcholine, cholesterol and alpha-tocopherol (4:3:0.07, mole %). Particle size, partition coefficient determination and in-vitro release studies were used to characterize the encapsulation process. Cytotoxicity was evaluated by the tetrazolium reduction test using sciatic nerve Schwann cells in culture. Local anaesthetic activity was assessed by mouse sciatic and rat infraorbital nerve blockades. Histological analysis was performed to verify the myotoxic effects evoked by RVC formulations. Plain (RVC(PLAIN)) and liposomal RVC (RVC(LUV)) samples were tested at 0.125%, 0.25% and 0.5% concentrations. Vesicle size distribution showed liposomal populations of 370 and 130 nm (85 and 15%, respectively), without changes after RVC encapsulation. The partition coefficient value was 132 +/- 26 and in-vitro release assays revealed a decrease in RVC release rate (1.5 fold, P < 0.001) from liposomes. RVC(LUV) presented reduced cytotoxicity (P < 0.001) when compared with RVC(PLAIN). Treatment with RVC(LUV) increased the duration (P < 0.001) and intensity of the analgesic effects either on sciatic nerve blockade (1.4-1.6 fold) and infraorbital nerve blockade tests (1.5 fold), in relation to RVC(PLAIN). Regarding histological analysis, no morphological tissue changes were detected in the area of injection and sparse inflammatory cells were observed in only one of the animals treated with RVC(PLAIN) or RVC(luv) at 0.5%. Despite the differences between these preclinical studies and clinical conditions, we suggest RVC(LUV) as a potential new formulation, since RVC is a new and safe local anaesthetic agent.

Increased sensory neuron apoptotic death 2 weeks after peripheral axotomy in C57BL/6J mice compared to A/J mice.

Peripheral nerve transection results in a disconnection of the neuron from its target. As a result, a series of metabolic changes occur in the cell body that may cause neuronal death, mainly by apoptotic mechanisms. Although neurons from neonatal animals are the most susceptible, peripheral, lesion-induced, neuronal loss also occurs in adults, and is particularly evident in mouse sensory neurons. However, differences in genetic background cause particular isogenic strains of mice to react unevenly to peripheral nerve lesion. In this work, we investigated the occurrence of apoptosis as well as the ultrastructural changes in the dorsal root ganglion sensory neurons and satellite cells of C57BL/6J and A/J mice 2 weeks after ipsilateral sciatic nerve transection at the mid-thigh level. C57BL/6J mice displayed a stronger sensory neuron chromatolytic reaction that resulted in an increased loss of neurons when compared with isogenic A/J mice (p<0.01). Additionally, most of the degenerating neurons displayed the classic features of apoptosis. These findings reinforced previous data obtained by the terminal-deoxynucleotidyl transferase nick-end labeling (TUNEL) technique.

Enhanced peripheral nerve regeneration by the combination of a polycaprolactone tubular prosthesis and a scaffold of collagen with supramolecular organization.

The purpose of this study was to investigate the influence of implanting collagen with a supramolecular organization on peripheral nerve regeneration, using the sciatic nerve tubulization technique. For this purpose, adult female Sprague Dawley rats were divided into five groups: (1) TP - sciatic nerve repaired with empty polyethylene tubular prothesis (n = 10), (2) TPCL - nerve repair with empty polycaprolactone (PCL) tubing (n = 8), (3) TPCLF - repair with PCL tubing filled with an implant of collagen with a supramolecular organization (n = 10), (4) AG - animals that received a peripheral nerve autograft (n = 8), and (5) Normal nerves (n = 8). The results were assessed by quantification of the regenerated fibers, nerve morphometry, and transmission electron microscopy, 60 days after surgery. Immunohistochemistry and polarization microscopy were also used to analyze the regenerated nerve structure and cellular elements. The results showed that the AG group presented a larger number of regenerated axons. However, the TPCL and TPCLF groups presented more compact regenerated fibers with a morphometric profile closer to normal, both at the tube midpoint and 2 mm distal to the prosthesis. These findings were reinforced by polarization microscopy, which indicated a better collagen/axons suprastructural organization in the TPCLF derived samples. In addition, the immunohistochemical results obtained using the antibody anti-p75NTR as a Schwann cell reactivity marker demonstrated that the Schwann cells were more reactive during the regenerative process in the TPCLF group as compared to the TPCL group and the normal sciatic nerve. Altogether, the results of this study indicated that the implant of collagen with a supramolecular organization positively influenced and stimulated the regeneration process through the nerve gap, resulting in the formation of a better morphologically arranged tissue.

Peripheral nerve regeneration through biodegradable conduits prepared using solvent evaporation.

The present study explored a new approach to the production of tubular conduits designed for peripheral nerve repair. Poly(L-lactic acid) (PLLA) and polycaprolactone (PCL) membranes were obtained after solvent evaporation and wrapped around a mandrel. The effectiveness of nerve regeneration was compared with that obtained with polyethylene and PCL extruded prostheses 30 and 60 days after surgery. The comparison between extruded and membrane-derived tubes clearly showed structural differences that were directly proportional to the hardness and transparency. An important factor to be considered is that the fiber count indicated that membrane-derived PCL tubes provided a significantly greater number of axons 30 days after repair. Sixty days after the procedure, the greatest regenerative performance was obtained with PCL, regardless of tube construction method. An intense imunolabeling of S100, type IV collagen, and laminin could be observed in the tissue obtained from membrane-derived PCL and PLLA groups, indicating that such constructs were able to positively stimulate Schwann cell responses. Overall, the results provided evidence that membrane-derived conduits are an alternative preparation method for tubular prostheses for peripheral nerve regeneration.

MHC class I upregulation is not sufficient to rescue neonatal alpha motoneurons after peripheral axotomy.

Associated with neuronal death, profound synaptic changes occur in the spinal cord during the apoptotic process triggered after axotomy in neonatal rats. With respect to this, the major histocompatibility complex of class I (MHC class I) has recently emerged as a new mechanism related to synaptic stripping and plasticity. The present study investigated the impact of upregulating MHC class I expression by treatment with beta interferon (beta INF) on motoneuron survival, synaptic plasticity and astrogliosis after neonatal sciatic nerve injury. P2 rats were subjected to unilateral axotomy followed by three days of beta INF treatment. The results were analyzed by counting Nissl stained motoneurons, immunohistochemistry (anti-synaptophysin, MHC class I, GFAP and Iba-1) and transmission electron microscopy. INF treatment induced an increased expression of MHC class I, which resulted in a stronger synaptic elimination process in the spinal cord, as seen by the synaptophysin labeling. GFAP and Iba-1 upregulation were not significantly altered by the INF treatment, displaying the same degree of enhanced reactivity as compared to the placebo group. The ultrastructural analysis showed that, apart from the overall reduction of inputs in the neuropil, no statistical differences were present when comparing the INF and placebo treated animals. Also, neuronal survival was not altered by cytokine administration. The present results provide evidence that MHC class I upregulation after neonatal injury does not change the fate of lesioned motoneurons. In this way, the lack of neurotrophic support may cause broader synaptic loss, which superposes the more subtle effects of the upregulation of MHC class I.

Peripheral nerve regeneration through the nerve tubulization technique

Transection of peripheral nerve results in a loss of function at the target organ that can rarely be recovered without surgical repais. Such an intervention usualy involves nerve autografting but is complicated by problems such as the need for secondary surgery, a limited donor nerve supply and loss of sensitivity in the donor nerve area. An alternative approach involving repair by nerve tubulization has been extensively used to study substances that may improve the regenerative process. An interesting feature of the tubulization technique is the possibility of filling the tube with substances that can enhance regeneration. Such substances include collagen, laminin, hyaluronic acid, fibronectin and more recently, glycosaminoglycans alone or with collagen. Biopolymers, purified glial cells, and neurothophic factors have also been tested. By using the tubulation technique, it has been possible to increase the number of regenerating fibers and the gap between the stumps. In this review, we discuss some of the basic concepts of this technique, as well as recent advances in this field.

Effects of aggrecan on schwann cell migration in vitro and nerve regeneration in vivo

Although the role of many small proteoglycans in regeneration of the nervous system has been established, little is known about the involvement of large proteoglycans. In this study, we evaluated the effects of aggrecan, a high molecular mass proteoglycan, on Schwann cells in vitro and investigated its effects on axonal regeneration after sciatic nerve tubulization. The number of regenerated axons and their morphometrical parameters were determined in vivo. Aggrecan increased the number and viability of Schwann cells in vitro. Similarly, the number of regenerated fibers increased significantly when aggrecan was applied in vivo, but there were no alterations in the morphometrical parameters. These results indicate that aggrecan contributes to the regeneration of peripheral axons and has a positive effect on the Schwann cells.