Quantitative and ultrastructural study of serotonin innervation of the globus pallidus in squirrel monkeys.

The present immunohistochemical study was aimed at characterizing the serotonin (5-HT) innervation of the internal (GPi) and external (GPe) pallidal segments in the squirrel monkey (Saimiri sciureus) with an antibody against the 5-HT transporter (SERT). At the light microscopic level, unbiased counts of SERT+ axon varicosities showed that the density of innervation is similar in the GPi (0.57 ± 0.03 × 10(6)  varicosities/mm(3) of tissue) and the GPe (0.60 ± 0.04 × 10(6) ), with the anterior half of both segments being more densely innervated than the posterior half. Dorsoventral and mediolateral decreasing gradients of SERT varicosities occur in both pallidal segments, but are statistically significant only in the GPi. The neuronal density being significantly greater in the GPe (3.41 ± 0.23 × 10(3)  neurons/mm(3) ) than in the GPi (2.90 ± 0.11 × 103), the number of 5-HT axon varicosities per pallidal neuron was found to be superior in the GPi (201 ± 27) than in the GPe (156 ± 26). At the electron microscopic level, SERT+ axon varicosities are comparable in size and vesicular content in GPi and GPe, where they establish mainly asynaptic contacts with unlabeled profiles. Less than 25% of SERT+ varicosities display a synaptic specialization, which is of the symmetrical or asymmetrical type and occurs exclusively on pallidal dendrites. No SERT+ axo-axonic synapses are present, suggesting that 5-HT exerts its well-established modulatory action upon various pallidal afferents mainly through diffuse transmission, whereas its direct control of pallidal neurons results from both volumic and synaptic release of the transmitter.

In vivo enhancement of sensory perception recovery in a tissue-engineered skin enriched with laminin.

The use of autologous reconstructed skin appears to be a promising treatment for the permanent coverage of deep and extensive burns. However, the capability of reconstructed skin transplanted on wounds to promote recovery of sensory perception is a major concern. Our aim was to assess the effect of laminin on cutaneous nerve regeneration. We prepared collagen-chitosan sponges enriched with 0, 1, 10 or 50 microg of laminin/sponge to produce tissue-engineered reconstructed skins by culture of human fibroblasts and keratinocytes, then grafted on the back of athymic mice for 120 days. Immunohistochemical studies demonstrated that there were 7 times more neurofilament 150 kD-positive nerve fibers migrating in the graft in the samples enriched with 10 microg laminin/sponge, compared to reconstructed skin without laminin, 120 days after graft. A significant improvement in the current perception threshold of the Abeta and Adelta nerve fibers was measured using a Neurometer in all grafts enriched with laminin. In addition, the type C nerve fibers reached an identical current perception threshold than mouse skin, in all reconstructed skins enriched or not with laminin. We conclude that the use of a tissue-engineered autologous skin graft enriched with laminin has the potential to efficiently optimize cutaneous sensory nerve regeneration in vivo.

Differentiation of human adult skin-derived neuronal precursors into mature neurons.

The isolation of autologous neuronal precursors from skin-derived precursor cells extracted from adult human skin would be a very efficient source of neurons for the treatment of various neurodegenerative diseases. The purpose of this study was to demonstrate that these neuronal precursors were able to differentiate into mature neurons. We isolated neuronal precursors from breast skin and expanded them in vitro for over ten passages. We showed that 48% of these cells were proliferating after the first passage, while this growth rate decreased after the second passage. We demonstrated that 70% of these cells were nestin-positive after the third passage, while only 17% were neurofilament M-positive after 7 days of differentiation. These neuronal precursors expressed betaIII tubulin, the dendritic marker MAP2 and the presynaptic marker synaptophysin after 7 days of in vitro maturation. They also expressed the postsynaptic marker PSD95 and the late neuronal markers NeuN and neurofilament H after 21 days of differentiation, demonstrating they became terminally differentiated neurons. These markers were still expressed after 50 days of culture. The generation of autologous neurons from an accessible adult human source opens many potential therapeutic applications and has a great potential for the development of experimental studies on normal human neurons.

Quantitative method to evaluate the functionality of the trigeminal nerve.

PURPOSE: The primary objective of this study was to yield normal range values, with a current perception threshold technique, that could be used to assess the functionality of the third division of the trigeminal nerve on a healthy population. Moreover, we wanted to evaluate the impact of gender and training on these values. PATIENTS AND METHODS: Standardized current perception threshold (CPT) measures using constant alternating current sinusoid waveform stimulus at 5, 250, and 2,000 HZ were obtained from 50 healthy patients at the mental foramen area bilaterally using a Neurometer current perception threshold device (Neurotron Inc, Baltimore, MD) (2,000 Hz specifically stimulates Abeta fibers, 250 Hz Adelta fibers, and 5 Hz stimulates C fibers). RESULTS: The mean CPT values for the 2,000, 250, and 5 Hz groups were respectively, 157.6 +/- 54.67, 53.10 +/- 27.64, and 33.44 +/- 23.17 mAmp. These values were used to construct a CPT scale to classify patients in the hyperesthetic, normative, or hypoesthetic range. There were no significant differences when comparing CPT values in men and women except in the 2,000 Hz group (P < .02; n = 23). In addition, the test was carried out a first time on the right side and a second time on the left. This training procedure showed a significant decrease in the CPT values in men at 2,000 Hz (P < .01; n = 23) for the second measure. CONCLUSION: The Neurometer can be beneficial for the evaluation and intraneural localization of sensory dysfunctions associated with the third division of the trigeminal nerve. It also could be used for initial diagnosis and subsequent evaluation of the patient's neurologic status through the course of their condition.

In vitro reconstruction of a tissue-engineered endothelialized bladder from a single porcine biopsy.

OBJECTIVE: Augmentation of the urinary bladder using a tissue-engineered approach with autologous cells is a very promising technique. To prevent risks of necrosis after transplantation, the graft vascularization process could be markedly enhanced by incorporation of autologous endothelial cells in the tissue-engineered organ. The purpose of this study was to develop a separation technique to extract four bladder cell types from the same biopsy, and to prepare an endothelialized reconstructed bladder. MATERIALS AND METHODS: Fibroblasts, smooth muscle cells (SMC), urothelial cells (UC) and endothelial cells (EC) were extracted from a small porcine bladder biopsy. The SMC, fibroblasts and EC were seeded on the top of the sponge and cultured for 10 days. Then, the UC were seeded on top of these cells for 15 additional days to produce a three-dimensional bladder wall. RESULTS: The UC and EC extracts from a single porcine biopsy were 97.2+/-0.6% keratin 8/18-positive and 97.7+/-0.3% PECAM-1-positive pure cells, respectively, as assessed by flow cytometry. The SMC could not be dissociated from fibroblasts, and were present as 37+/-0.5% desmin-positive cells. UC differentiated into a urothelium characterized by umbrella cells and a laminin-positive basal membrane. The EC reorganized in the matrix to form PECAM-1-positive capillary-like tubes. CONCLUSION: This new model of tissue-engineered bladder has the main advantages of being at least 2mm thick, autologous, and able to promote the formation of capillary-like tubes. It could be a promising alternative to the use of gastrointestinal segments to improve bladder capacity.