Francesco Langone

Peripheral nerve repair using a poly(organo)phosphazene tubular prosthesis

Nerve regeneration experiments were carried out using tubular nerve guides of poly[(ethylalanato)1.4(imidazolyl)0.6phosphazene] (PEIP). By means of in vivo tests, this polymer was found to be biodegradable and transformed into harmless products. The tubular nerve guides were prepared by deposition of the dissolved polymer on a glass capillary tube, followed by evaporation of the solvent (methylene dichloride). After transectioning, rat sciatic nerve stumps were immediately sutured into the ends of 10-mm-long polymer tubes. On removal of the prosthesis, after implantation for 45 d, a tissue cable was found bridging the nerve stumps in all cases. Histological analysis revealed that the tissue cable was essentially composed of a regenerated nerve fibre bundle. A parallel series of experiments was undertaken to compare the use of silicone tubes that are not biodegradable and are most frequently used for studies of nerve regeneration with tubulization techniques. The advantages of biodegradable PEIP tubular nerve guides used for peripheral nerve repair are discussed.

Bone marrow stromal cells and resorbable collagen guidance tubes enhance sciatic nerve regeneration in mice

We evaluated peripheral nerve regeneration using a tubular nerve guide of resorbable collagen filled with either bone marrow-derived cells (BMDCs) in Dulbeccos cell culture medium (DMEM) or with DMEM alone (control). The control group received just the culture medium (vehicle). The left sciatic nerves of ten isogenic mice were transected and the tubular nerve guides were sutured to the end of the proximal and distal nerve stumps. Motor function was tested at 2, 4 and 6 weeks after surgery using the walking track test. The pawprints were analyzed and the print lengths (PL) were measured to evaluate functional recovery. After 6 weeks, mice were anesthetized, perfused transcardially with fixative containing aldehydes, and the sciatic nerves and tubes were dissected and processed for scanning and transmission electron microscopy. Scanning electron microscopy of the collagen tube revealed that the tube wall became progressively thinner after surgery, proving that the tube can be resorbed in vivo. Quantitative analysis of the regenerating nerves showed that the number of myelinated fibers and the myelin area were significantly increased in the experimental group. Also, motor function recovery was faster in animals that received the cell grafts. These results indicate that the collagen tube filled with BMDCs provided an adequate and favorable environment for the growth and myelination of regenerating axons compared to the collagen tube alone.

THE ANTIDEPRESSIVE EFFECT OF THE PHYSICAL EXERCISE CORRELATES WITH INCREASED LEVELS OF MATURE BDNF, AND proBDNF PROTEOLYTIC CLEAVAGE-RELATED GENES, p11 AND tPA

Clinical studies show an evident antidepressive effect of physical exercise and animal research corroborate such evidence. However, the neurobiological mechanisms underlying the antidepressive effect of exercise are not completely understood. Notwithstanding, it is known that exercise increases brain-derived neurotrophic factor (BDNF) expression in the hippocampus similarly to antidepressant drugs. BDNF is synthesized as a precursor molecule that undergoes a proteolytic cleavage to generate either a mature or a truncated isoform. Precursor and mature BDNF are assumed to elicit opposing biological effects in neuroplasticity. In the present study we investigated the effect of voluntary physical activity on precursor and mature brain-derived neurotrophic factor levels and on proBDNF cleavage related genes, p11 and tissue plasminogen activator (tPA), as well as the antidepressive and cognitive effects of voluntary physical activity. Mice had access to mobile or locked running wheels for 28 days and were submitted to forced-swim, tail suspension and water maze tests. Their hippocampi were dissected and analyzed by Western blot and real time RT-PCR. Voluntary physical activity, but not locked wheel exposure, induced a robust increase in hippocampal mature BDNF protein levels, as well as in p11 and tPA mRNA expression; and also promoted antidepressive effects and improved learning, when compared with sedentary mice. On the other hand, there were no significant differences between any groups in the expression of precursor or truncated isoforms of BDNF. Our data suggest that the antidepressive effect of the physical exercise may depend, at least in part, on changes in BDNF post-translational processing.

Physical Exercise and Antidepressants Enhance BDNF Targeting in Hippocampal CA3 Dendrites: Further Evidence of a Spatial Code for BDNF Splice Variants

Brain-derived neurotrophic factor (BDNF) is encoded by multiple BDNF transcripts, whose function is unclear. We recently showed that a subset of BDNF transcripts can traffic into distal dendrites in response to electrical activity, while others are segregated into the somatoproximal domains. Physical exercise and antidepressant treatments exert their beneficial effects through upregulation of BDNF, which is required to support survival and differentiation of newborn dentate gyrus (DG) neurons. While these DG processes are required for the antidepressant effect, a role for CA1 in antidepressant action has been excluded, and the effect on CA3 neurons remains unclear. Here, we show for the first time that physical exercise and antidepressants induce local increase of BDNF in CA3. Voluntary physical exercise for 28 consecutive days, or 2-week treatment with 10 mg/kg per day fluoxetine or reboxetine, produced a global increase of BDNF mRNA and protein in the neuronal somata of the whole hippocampus and a specific increase of BDNF in dendrites of CA3 neurons. This increase was accounted for by BDNF exon 6 variant. In cultured hippocampal neurons, application of serotonin or norepinephrine (10–50 μM) induced increase in synaptic transmission and targeting of BDNF mRNA in dendrites. The increased expression of BDNF in CA3 dendrites following antidepressants or exercise further supports the neurotrophin hypothesis of antidepressants action and confirms that the differential subcellular localization of BDNF mRNA splice variants provides a spatial code for a selective expression of BDNF in specific subcellular districts. This selective expression may be exploited to design more specific antidepressants.

Neuroprotective action of melatonin on neonatal rat motoneurons after sciatic nerve transection

The neuronal isoform of nitric oxide synthase (nNOS), a NADPH-dependent diaphorase, is considered to play a role in motoneuron death induced by sciatic nerve transection in neonatal rats. Neuronal loss in these circumstances has been correlated with nitric oxide (NO) production and NADPH-diaphorase positivity in motoneurons after axotomy. In the present study we looked for a possible protective effect of melatonin, an antioxidant agent and inhibitor of nNOS, on spinal motoneurons after axonal injury. Neonatal Wistar rats (P2) were submitted to sciatic nerve transection and allowed to survive to P7. Melatonin at doses of 1, 5, 10, 50 and 100 mg/kg was given subcutaneously before and at intervals after the surgery. Controls operated in the same way received dilution vehicle or no treatment. The animals were killed by perfusion of fixative and the spinal cord was examined in serial paraffin sections. The motoneurons of the sciatic pool were counted in the axotomized and contralateral sides. Immunohistochemistry for nNOS and glial fibrillary acidic protein was used to evaluate nNOS expression in the axotomized cells and the astrocytic response. We found that melatonin at doses of 1-50 mg/kg decreased neuronal death. Astrocytic hypertrophy in melatonin treated animals was less intense. There were no differences in nNOS expression between treated and control rats, and surviving motoneurons of the sciatic pool did not express the enzyme, suggesting that nNOS may not be involved in neuronal death or survival in these experimental conditions. Possible mechanisms of melatonin neuroprotection, which was equally effective at doses of 1-50 mg/kg, are discussed. Doses of 50 and 100 mg/kg caused failure to thrive, seizures or death. The fact that neuroprotective doses were far smaller than toxic ones should encourage testing of melatonin in neurologic diseases.

Enhancement of sciatic nerve regeneration after vascular endothelial growth factor (VEGF) gene therapy

F. R. Pereira Lopes, B. C. G. Lisboa, F. Frattini, F. M. Almeida, M. A. Tomaz, P. K. Matsumoto, F. Langone, S. Lora, P. A. Melo, R. Borojevic, S. W. Han and A. M. B. Martinez (2011) Neuropathology and Applied Neurobiology37, 600–612

The essential amino acid lysine acts as precursor of glutamate in the mammalian central nervous system

Lysine has long been recognized as an essential amino acid for humans and the lack or low supply of this compound in the diet may lead to mental and physical handicaps. Since lysine is severely restricted in cereals, the most important staple food in the world, the understanding of its biological roles must be a major concern. Here we show that lysine is an important precursor for de novo synthesis of glutamate, the most significant excitatory neurotransmitter in the mammalian central nervous system. We also show that the synthesis of glutamate from lysine, which is carried out by the saccharopine pathway, is likely to take place in neurons.

Neuronal degeneration and gliosis time-course in the mouse hippocampal formation after pilocarpine-induced status epilepticus.

Temporal lobe epilepsy (TLE) is the most common type of human epilepsy and has been related with extensive loss of hippocampal pyramidal and dentate hilar neurons and gliosis. Many characteristics of TLE are reproduced in the pilocarpine model of epilepsy in mice. This study analyzed the neuronal damage, assessed with Fluoro-Jade (FJB) and cresyl violet, and gliosis, investigated with glial fibrilary acidic protein (GFAP) immunohistochemistry, occurring in the hippocampal formation of mice at 3, 6, 12 and 24h, 1 and 3 weeks after the pilocarpine-induced status-epilepticus (SE) onset. The maximum neuronal damage score and the FJB-positive neurons peak were found in the hilus of dentate gyrus 3 and 12 h after SE onset (P<0.05), respectively. At 1 week after SE onset, the greatest neuronal damage score was detected in the CA1 pyramidal cell layer and the greatest numbers of FJB-positive neurons were found both in the CA1 and CA3 pyramidal cell layers (P<0.05). The molecular, CA3 and CA1 pyramidal cell layers expressed highest presence of GFAP immunoreaction at 1 and 3 weeks after SE onset (P<0.05). Our findings show that, depending on the affected area, neuronal death and gliosis can occur within few hours or weeks after SE onset. Our results corroborate previous studies and characterize short time points of temporal evolution of neuropathological changes after the onset of pilocarpine-induced SE in mice and evidences that additional studies of this temporal evolution may be useful to the comprehension of the cellular mechanisms underlying epileptogenesis.

Apoptosis of spinal interneurons induced by sciatic nerve axotomy in the neonatal rat is counteracted by nerve growth factor and ciliary neurotrophic factor

We have previously shown that not only motoneurons and dorsal root ganglion cells but also small neurons, presumably interneurons in the spinal cord, may undergo apoptotic cell death as a result of neonatal peripheral nerve transection in the rat. With the aid of electron microscopy, we have here demonstrated that apoptosis in the spinal cord is confined to neurons and does not involve glial cells at the survival time studied (24 hours). To define the relative importance of the loss of a potential target (motoneuron) and a potential afferent input (dorsal root ganglion cell) for the induction of apoptosis in interneurons in this situation, we have compared the distributions and time courses for TUNEL labeling, which detects apoptotic cell nuclei, in the L5 segment of the spinal cord and the L5 dorsal root ganglion after sciatic nerve transection in the neonatal (P2) rat. In additional experiments, we studied the effects on TUNEL labeling of interneurons after treatment of the cut sciatic nerve with either ciliary neurotrophic factor (CNTF) to rescue motoneurons or nerve growth factor (NGF) to rescue dorsal root ganglion cells. The time courses of the TUNEL labeling in motoneurons and interneurons induced by the lesion show great similarities (peak at 8–48 hours postoperatively), whereas the labeling in dorsal root ganglion cells occurs later (24–72 hours). Both CNTF and NGF decrease the number of TUNEL‐labeled interneurons, but there is a regional difference, in that CNTF preferentially saves interneurons in deep dorsal and ventral parts of the spinal cord, whereas the rescuing effects of NGF are seen mainly in the superficial dorsal horn. The results are interpreted as signs of a trophic dependence on both the target and the afferent input for the survival of interneurons neonatally. J. Comp. Neurol. 447:381–393, 2002.

Ciliary neurotrophic factor (CNTF) signals through STAT3–SOCS3 pathway and protects rat pancreatic islets from cytokine-induced apoptosis

CNTF is a cytokine that promotes survival and/or differentiation in many cell types, including rat pancreatic islets. In this work, we studied the mechanism of CNTF signal in neonatal rats pancreatic islets isolated by the collagenase method and cultured for 3 days in RPMI medium without (CTL) or with 1 nM of CNTF. The medium contained, when necessary, specific inhibitors of the PI3K, MAPK and JAK/STAT3 pathways. mRNA expression (RT-PCR) and protein phosphorylation (Western blot) of Akt, ERK1/2 and STAT3, and SOCS-3 (RT-PCR and Western blot), as well as glucose-stimulated insulin secretion (GSIS) (Radioimmunoassay), were analyzed. Our results showed that Akt, ERK1 and STAT3 mRNA expression, as well as phosphorylated Akt and ERK1/2, was not affected by CNTF treatment. CNTF increased cytoplasmatic and nuclear phosphorylated STAT3, and the SOCS3 mRNA and protein expression. In addition, CNTF lowered apoptosis and impaired GSIS. These effects were blocked by the JAK inhibitor, AG490 and by the STAT3 inhibitor Curcumin, but not by the MAPK inhibitor, PD98059, nor by the PI3K inhibitor, Wortmannin. In conclusion, CNTF signals through the JAK2/STAT3 cascade, increases SOCS3 expression, impairs GSIS and protects neonatal pancreatic rat islets from cytokine-induced apoptosis. These findings indicate that CNTF may be a potential therapeutic tool against Type 1 and/or Type 2 diabetes.