Aberrant perineuronal nets alter spinal circuits, impair motor function, and increase plasticity.

Perineuronal nets (PNNs) are a specialized extracellular matrix that have been extensively studied in the brain. Cortical PNNs are implicated in synaptic stabilization, plasticity inhibition, neuroprotection, and ionic buffering. However, the role of spinal PNNs, mainly found around motoneurons, is still unclear. Thus, the goal of this study is to elucidate the role of spinal PNNs on motor function and plasticity in both intact and spinal cord injured mice. We used transgenic mice lacking the cartilage link protein 1 (Crtl1 KO mice), which is implicated in PNN assembly. Crtl1 KO mice showed disorganized PNNs with an altered proportion of their components in both motor cortex and spinal cord. Behavioral and electrophysiological tests revealed motor impairments and hyperexcitability of spinal reflexes in Crtl1 KO compared to WT mice. These functional outcomes were accompanied by an increase in excitatory synapses around spinal motoneurons. Moreover, following spinal lesions of the corticospinal tract, Crtl1 KO mice showed increased contralateral sprouting compared to WT mice. Altogether, the lack of Crtl1 generates aberrant PNNs that alter excitatory synapses and change the physiological properties of motoneurons, overall altering spinal circuits and producing motor impairment. This disorganization generates a permissive scenario for contralateral axons to sprout after injury.

Effective decellularization of human nerve matrix for regenerative medicine with a novel protocol.

Injuries to the peripheral nerves represent a frequent cause of permanent disability in adults. The repair of large nerve lesions involves the use of autografts, but they have several inherent limitations. Overcoming these limitations, the use of decellularized nerve matrix has emerged as a promising treatment in tissue regenerative medicine. Here, we generate longer human decellularized nerve segments with a novel decellularization method, using nonionic, zwitterionic, and enzymatic incubations. Efficiency of decellularization was measured by DNA quantification and cell remnant analysis (myelin, S100, neurofilament). The evaluation of the extracellular matrix (collagen, laminin, and glycosaminoglycans) preservation was carried out by enzyme-linked immunosorbent assay (ELISA) or biochemical methods, along with histological and immunofluorescence analysis. Moreover, biomechanical properties and cytocompatibility were tested. Results showed that the decellularized nerves generated with this protocol have a concentration of DNA below the threshold of 50 ng/mg of dry tissue. Furthermore, myelin, S100, and MHCII proteins were absent, although some neurofilament remnants could be observed. Moreover, extracellular matrix proteins were well maintained, as well as the biomechanical properties, and the decellularized nerve matrix did not generate cytotoxicity. These results show that our method is effective for the generation of decellularized human nerve grafts. The generation of longer decellularized nerve segments would allow the understanding of the regenerative neurobiology after nerve injuries in both clinical assays and bigger animal models. Effective decellularization of human nerve matrix for regenerative medicine with a novel protocol. Combination of zwitterionic, non-ionic detergents, hyperosmotic solution and nuclease enzyme treatment remove cell remnants, maintain collagen, laminin and biomechanics without generating cytotoxic leachables.

Voluntary wheel running preserves lumbar perineuronal nets, enhances motor functions and prevents hyperreflexia after spinal cord injury.

Perineuronal nets (PNN) are a promising candidate to harness neural plasticity since their activity-dependent modulation allows to either stabilize the circuits or increase plasticity. Modulation of plasticity is the basis of rehabilitation strategies to reduce maladaptive plasticity after spinal cord injuries (SCI). Hence, it is important to understand how spinal PNN are affected after SCI and rehabilitation. Thus, this work aims to describe functional and PNN changes after thoracic SCI in mice, followed by different activity-dependent therapies: enriched environment, voluntary wheel and forced treadmill running. We found that the contusion provoked thermal hyperalgesia, hyperreflexia and locomotor impairment as measured by thermal plantar test, H wave recordings and the BMS score of locomotion, respectively. In the spinal cord, SCI reduced PNN density around lumbar motoneurons. In contrast, activity-based therapies increased motoneuron activity and reversed PNN decrease. The voluntary wheel group showed full preservation of PNN which also correlated with reduced hyperreflexia and better locomotor recovery. Furthermore, both voluntary wheel and treadmill running reduced hyperalgesia, but this finding was independent of lumbar PNN levels. In the brainstem sensory nuclei, SCI did not modify PNN whereas some activity-based therapies reduced them. The results of the present study highlight the impact of SCI on decreasing PNN at caudal segments of the spinal cord and the potential of physical activity-based therapies to reverse PNN disaggregation and to improve functional recovery. As modulating plasticity is crucial for restoring damaged neural circuits, regulating PNN by activity is an encouraging target to improve the outcome after injury.

Tubulization with chitosan guides for the repair of long gap peripheral nerve injury in the rat.

Biosynthetic guides can be an alternative to nerve grafts for reconstructing severely injured peripheral nerves. The aim of this study was to evaluate the regenerative capability of chitosan tubes to bridge critical nerve gaps (15 mm long) in the rat sciatic nerve compared with silicone (SIL) tubes and nerve autografts (AGs). A total of 28 Wistar Hannover rats were randomly distributed into four groups (n = 7 each), in which the nerve was repaired by SIL tube, chitosan guides of low (∼2%, DAI) and medium (∼5%, DAII) degree of acetylation, and AG. Electrophysiological and algesimetry tests were performed serially along 4 months follow-up, and histomorphometric analysis was performed at the end of the study. Both groups with chitosan tubes showed similar degree of functional recovery, and similar number of myelinated nerve fibers at mid tube after 4 months of implantation. The results with chitosan tubes were significantly better compared to SIL tubes (P < 0.01), but lower than with AG (P < 0.01). In contrast to AG, in which all the rats had effective regeneration and target reinnervation, chitosan tubes from DAI and DAII achieved 43 and 57% success, respectively, whereas regeneration failed in all the animals repaired with SIL tubes. This study suggests that chitosan guides are promising conduits to construct artificial nerve grafts.

Schwann cell targeting via intrasciatic injection of AAV8 as gene therapy strategy for peripheral nerve regeneration.

Efficient transduction of the peripheral nervous system (PNS) is required for gene therapy of acquired and inherited neuropathies, neuromuscular diseases and for pain treatment. We have characterized the tropism and transduction efficiency of different adeno-associated vectors (AAV) pseudotypes after sciatic nerve injection in the mouse. Among the pseudotypes tested, AAV2/1 transduced both Schwann cells and neurons, AAV2/2 infected only sensory neurons and AAV2/8 preferentially transduced Schwann cells. AAV2/8 expression in the sciatic nerve was detected up to 10 weeks after administration, the latest time point analyzed. The injected mice developed neutralizing antibodies against all AAVs tested; the titers were higher against AAV2/1 than AAV2/2 and were the lowest for AAV2/8, correlating with a higher transgene expression overtime. AAV2/8 coding for ciliary neurotrophic factor (CNTF) led to an upregulation of P0 and PMP22 myelin proteins, four weeks after transduction of injured sciatic nerves. Importantly, CNTF-transduced mice showed a significant increase in both GAP43 expression in sensory neurons, a marker of axonal regeneration, and the compound muscle action potential. These results prove the utility of AAV8 as a gene therapy vector for Schwann cells to treat myelin disorders or to improve nerve regeneration.

Rolipram-induced elevation of cAMP or chondroitinase ABC breakdown of inhibitory proteoglycans in the extracellular matrix promotes peripheral nerve regeneration.

The inhibitory growth environment of myelin and extracellular matrix proteoglycans in the central nervous system may be overcome by elevating neuronal cAMP or degrading inhibitory proteoglycans with chondroitinase ABC (ChABC). In this study, we asked whether similar mechanisms operate in peripheral nerve regeneration where effective Wallerian degeneration removes myelin and extracellular proteoglycans slowly. We repaired transected common peroneal (CP) nerve in rats and either elevated cAMP in the axotomized neurons by subcutaneous rolipram, a specific inhibitor of phosphodiesterase IV, and/or promoted degradation of proteoglycans in the distal nerve stump by local ChABC administration. Rolipram treatment significantly increased the number of motoneurons that regenerated axons across the repair site at 1 and 2 weeks, and increased the number of sensory neurons that regenerated axons across the repair site at 2 weeks. Local application of ChABC had a similar effect to rolipram treatment in promoting motor axon regeneration, the effect being no greater when rolipram and ChABC were administered simultaneously. We conclude that blocking inhibitors of axon regeneration by elevating cAMP or degrading proteoglycans in the distal nerve stump promotes peripheral axon regeneration after surgical repair of a transected nerve. It is likely that elevated cAMP is sufficient to encourage axon outgrowth despite the inhibitory growth environment such that simultaneous enzymatic proteoglycan degradation does not promote more axon regeneration than either elevated cAMP or proteoglycan degradation alone.

[Immunophilins: neuroprotective agents and promoters of neural regeneration]. / Las inmunofilinas: agentes neuroprotectores y promotores de la regeneración neural.

Immunophilins are a family of proteins mainly known because they act as receptors of the immunosuppressant drugs cyclosporin A (CsA) and FK506. Immunophilins serve several general functions, including regulation of mitochondrial permeability, modulation of ion channels stability and acting as chaperones for a variety of proteins. However, immunophilins are also present at high density in the nervous system. CsA, FK506 and other derivatives inhibit the function of immunophilins and, through bloking or activating several intracellular pathways, it has been shown that they exert neuroprotective effects in different experimental models of ischemia, Parkinson's disease and excitotoxic insults. Moreover, FK506 also has neuroregenerative effects, by enhancing the axonal regeneration rate after lesions of the peripheral nervous system. The development of new agents that selectively bind to immunophilins opens new interesting perspectives for the therapy of degenerative diseases and injuries of the nervous system.

Las inmunofilinas: agentes neuroprotectores y promotores de la regeneración neural / Immunophilins: neuroprotective agents and promoters of neural regeneration

Las inmunofilinas son una familia de proteínas principalmente conocidas porque actúan como receptores de los fármacos inmunodepresores ciclosporina A (CsA) y FK506. Las inmunofilinas ejercen diversas funciones generales, que incluyen las de regular la permeabilidad mitocondrial, modular la estabilidad de canales iónicos y actuar como chaperonas para una variedad de proteínas. Sin embargo, las inmunofilinas se encuentran en particular abundancia en el sistema nervioso. La CsA, el FK506 y otros derivados inhiben la función de las inmunofilinas y, a través del bloqueo o activación de diversos procesos intracelulares, se ha demostrado que ejercen efectos neuroprotectores en distintos modelos experimentales de isquemia cerebral, enfermedad de Parkinson y lesiones excitotóxicas. Asimismo, el FK506 también tiene efectos neurorregeneradores, acelerando la velocidad de regeneración axonal tras lesiones del sistema nervioso periférico. El desarrollo farmacológico de nuevos agentes ligandos selectivos de inmunofilinas ofrece nuevas perspectivas de interés en el tratamiento de los procesos degenerativos y las lesiones del sistema nervioso. (AU)

Effects of FK506 on nerve regeneration and reinnervation after graft or tube repair of long nerve gaps.

We compared the effects of FK506 administration on regeneration and reinnervation after sciatic nerve resection and repair with an autologous graft or with a silicone tube leaving a 6-mm gap in the mouse. Functional reinnervation was assessed by noninvasive methods to determine recovery of motor, sensory, and sweating functions in the hindpaw over 4 months after operation. Morphometric analysis of the regenerated nerves was performed at the end of follow-up. The nerve graft allowed for faster and higher levels of reinnervation in the four functions tested than silicone tube repair. Treatment with FK506 (for the first 9 weeks only) resulted in a slight, although not significant, improvement of the onset of reinnervation and of the maximal degree of recovery achieved after autografting. The recovery of pain sensibility and of the compound nerve action potentials in the digital nerves, which directly depend on axonal regeneration, showed better progression with FK506 than reinnervation of muscles and sweat glands, which require reestablishment of synaptic contacts with target cells. The myelinated fibers in the regenerated nerve showed a more mature appearance in the FK506-treated rats. However, FK506 showed a marginal effect in situations in which regeneration was limited, as in a silicone tube bridging a 6-mm gap in the mouse sciatic nerve. In conclusion, treatment with FK506 improved the rate of functional recovery after nerve resection and autograft repair.

[Chylothorax. An infrequent complication in surgical exeresis of the esophagus]. / Quilotórax. Una complicación poco frecuente en la cirugía exerética de esófago.

We report a case of massive bilateral chylothorax occurring after surgical resection of the esophagus in a patient with esophageal neoplasm. The surgical approach consisted of a thoracotomy and a cervicostomy. The relevance of this case is based on the low incidence of chylothorax after esophageal surgery. The literature indicates an incidence of about 0.9 to 3%. In our series of 200 patients operated on during the last 10 years we found and incidence of 2%. The patient herein reported presented an immediate postoperative clinical picture of respiratory insufficiency associated with the presence of milky fluid in the pleural drainage. Hematologic and biochemical examination of the pleural fluid confirmed the diagnosis of chylothorax. During the first 24 hours we collected a total volume of 2,500 ml of chyle. The patient was treated with conservative procedures including parenteral nutrition and intravenous reposition of fluids. After 48 hours the total volume of chyle reached 8,500 ml and the patient died.