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Publisher Summary Amyotrophic lateral sclerosis (ALS) is characterized by the atrophy and death of the affected motoneurons with a marked dissolution of the nuclei and cytoplasm. This chapter considers three different aspects of ALS—the mechanism leading to cell death, the selective loss of motor neurons, and the protracted course of motoneuron degeneration. One of the common hallmarks in several motoneuron degenerative diseases, including sporadic and familial ALS, infantile spinal muscular atrophy, and hereditary sensory motor neuropathy, is the aberrant accumulation of neurofilaments (NF) in the affected motoneurons. Neurotrophic factors (NTF) play a critical role in supporting survival and differentiation of various neuronal population. Motoneurons respond to a variety of NTFs, namely insulin-like growth factor (IGF)-1, brain-derived neurotrophic factor, and neurturin. Neuronal growth factors hold promise for the treatment of neurodegenerative diseases. The presence of the blood-brain barrier, however, represents a major hurdle for the delivery of neurotrophic factors to the central nervous system (CNS). A technique involving the intrathecal implantation of polymer encapsulated cell-lines genetically engineered to release neurotrophic factors provides a means to continuously deliver neurotrophic factors directly within the CNS.

Intrathecal implants of adrenal chromaffin cells are known to release analgesic substances such as catecholamines and opioid peptides. In the present study, bovine chromaffin cells were encapsulated in a permselective polymer membrane which protects the cells from the host immune system and allows grafting of xenogeneic cells without immunosuppression. The effects of such implants were evaluated on the pain behavior resulting from a chronic constrictive injury (CCI) of the rat sciatic nerve. Sprague-Dawley rats with a unilateral lesion were implanted in the lumbar subarachnoid space and tested for mechanical/thermal allodynia and hyperalgesia. A significant reduction in pain was observed after mechanical non-nociceptive stimulation in animals implanted with chromaffin cells. Furthermore, these animals showed decreased signs of spontaneous pain. However, response to thermal non-noxious stimuli or to painful mechanical stimuli was not significantly decreased. Abundant clusters of viable chromaffin cells intensely labeled with the anti-tyrosine hydroxylase antibodies were observed in the retrieved implants. These results establish the analgesic efficacy of intrathecal encapsulated chromaffin cells in a chronic pain model of nerve injury. Immunoprotected allo- or xenogeneic chromaffin cells acting as 'mini pumps' continuously delivering neuroactive substances could be a useful therapy for patients suffering from neuropathic pain.

To determine whether neurturin (NTN), a recently identified homologue of glial cell line-derived neurotrophic factor (GDNF), is able to preserve tyrosine hydroxylase immunoreactivity (TH-IR) in a rat model of Parkinson's disease, polymer encapsulated cells genetically engineered to release NTN were implanted near the substantia nigra 1 week before a unilateral medial forebrain bundle axotomy. Animals were allowed to survive for 1 week post-axotomy. Upon sacrifice, animals that received a NTN capsule had a significantly higher percentage of TH-IR (lesioned side vs non-lesioned side) than animals that had received a capsule containing non-transfected parent cells. However, in contrast to GDNF, no reduction of turning was observed upon amphetamine rotation with NTN. Nevertheless, these results suggest that NTN might have a therapeutic value for the treatment of Parkinson's disease.

Brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF), members of distinct families of polypeptide growth factors, have been shown to support motoneurons under various in vitro and in vivo conditions. We used a model of motoneuron cell death induced by sciatic nerve section in newborn rats and compared the efficacy of BDNF and GDNF administered alone or simultaneously in order to determine whether combinations of neurotrophic proteins can produce more potent motoneuron rescue than individual factors. The factors were administered by different methods, including (i) a single dose on to the transected nerve, (ii) continuous delivery from implanted slow-release polymer rods (BDNF) or encapsulated cells (GDNF), and (iii) repeated systemic injections (BDNF). Irrespective of the method of administration, either factor alone produced rescue effects which dramatically declined at two weeks as compared to one week post-lesion. In contrast, this decrease was significantly reduced when BDNF and GDNF were used simultaneously provided that one factor was applied on to the nerve while the other was continuously released from the rods or capsules. Other combinations in which GDNF was replaced by ciliary neurotrophic factor or axokine-1 failed to reproduce such additive activity. Two conclusions can be made from these experiments. First, when BDNF and GDNF are administered simultaneously but by distinct routes of delivery, their survival-promoting effects on the injured developing motoneurons are potentiated; second, even continuous delivery of each of these trophic factors alone cannot completely abrogate the time-dependent decline in rescue effects in this model of motoneuron cell death.

Glial cell line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor which has been purified on the basis of its ability to promote the survival of dopaminergic neurons in vitro. GDNF has subsequently been cloned and its sequence shown to be distantly related to transforming growth factor-beta (TGF-beta). To identify GDNF expressing cells in the adult rat brain, in situ hybridization using a digoxygenin (DIG)-labelled riboprobe has been performed. Our results show that GDNF mRNA is mainly expressed in neurons and that its synthesis is not restricted to dopaminergic areas. It is widely expressed in the cortex, the hippocampus, the striatum, the substantia nigra, the thalamus, the cerebellum and the spinal cord. Neuronal GDNF expression varies among brain regions as determined by the intensity of the in situ signal. Double labelling of the substantia nigra using tyrosine hydroxylase immunohistochemistry, associated with GDNF in situ hybridization, show that the majority of dopaminergic neurons express GDNF. The widespread expression of GDNF throughout the adult brain suggests that its administration in Parkinson's disease should be restricted to the altered structures, in order to avoid possible deleterious side effects.

Publisher Summary Neurotrophic factors may be of therapeutic importance for the treatment of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and Parkinson's disease (PD). The transplantation of polymer-encapsulated cells genetically engineered to continuously release neurotrophic factors at a relatively low dose is an extremely useful tool. Cell lines genetically engineered to release the neurotrophic factors CNTF or glial cell line-derived neurotrophic factor (GDNF) have been constructed. The capacity of GDNF to slow down the degeneration of dopaminergic neurons in a rat model of PD has been investigated. The results indicate that the continuous release of low levels of GDNF close to the substantia nigra is capable of protecting nigral dopaminergic neurons from axotomy-induced cell death and improving amphetamine-induced rotation by a mechanism other than dopaminergic striatal reinnervation. A phase I clinical trial using intrathecal delivery of human ciliary neurotrophic factor (CNTF) has been performed in six patients suffering from ALS. The study indicates that significant doses of CNTF can be delivered directly into the central nervous system, and that, in contrast to the side effects associated with systemic delivery of CNTF in ALS patients, no limiting adverse effects are observed in any of the patients exposed to low intrathecal doses of CNTF.

Parkinson’s disease (PD) is characterized by the progressive loss of the substantia nigra (SN) dopaminergic neurons projecting to the striatum. Neurotrophic factors may have the potential to prevent or slow down the degenerative process occurring in PD. To that end, we examined whether low amounts of glial cell line-derived neurotrophic factor (GDNF) continuously released from polymer-encapsulated genetically engineered cells are able to prevent the loss of tyrosine hydroxylase immunoreactivity (TH-IR) in SN neurons and ameliorate the amphetamine-induced rotational asymmetry in rats that have been subjected to a unilateral medial forebrain bundle (MFB) axotomy. Baby hamster kidney (BHK) cells transfected with the cDNA for GDNF were encapsulated in a polymer fiber and implanted unilaterally at a location lateral to the MFB and rostral to the SN. ELISA assays before implantation show that the capsules release ∼5 ng of GDNF/capsule per day. One week later, the MFB was axotomized unilaterally ipsilateral to the capsule placement. Seven days later, the animals were tested for amphetamine-induced rotational asymmetry and killed. The striatum was excised and analyzed either for catecholamine content or TH-IR, while the SN was immunostained for the presence of TH-IR. GDNF did not prevent the loss of dopamine in the striatum. However, GDNF significantly rescued TH-IR neurons in the SN pars compacta. Furthermore, GDNF also significantly reduced the number of turns per minute ipsilateral to the lesion under the influence of amphetamine. Improvement of rotational behavior in the absence of dopaminergic striatal reinnervation may reflect neuronal plasticity in the SN, as suggested by the dendritic sprouting observed in animals receiving GDNF. These results illustrate that the continuous release of low levels of GDNF close to the SN is capable of protecting the nigral dopaminergic neurons from an axotomy-induced lesion and significantly improving pharmacological rotational behavior by a mechanism other than dopaminergic striatal reinnervation.

Background Tau related pathology is the most reliable predictor of cognitive decline in Alzheimer’s disease. However, it remains unclear by which mechanisms tau contributes to neurodegeneration and neuronal dysfunction. The use of viral vectors provides an effective approach to replicate cardinal features of tauopathies in the mouse brain. AAV vectors were designed for overexpression of various forms of human tau, in order to dissect the mechanisms underlying tau hyperphosphorylation, aggregation and neurotoxicity in vivo.

Glial cell line-derived neurotrophic factor (GDNF), a member of the TG F-beta superfamily, has been shown to be a highly potent neurotrophic factor that enhances survival of various neuronal cell types including motoneurons. To assess its therapeutic potential in treating neurodegenerative diseases such as amyotrophic lateral sclerosis, we treated mutant mice displaying motoneuron degeneration (progressive motor neuropathy; pmn) with encapsulated GDNF-secreting cells. Effects of GDNF treatment on pmn/pmn mice were compared with previous results obtained with ciliary neurotrophic factor (CNTF) [Sagot Y, Tan SA, Baetge E, Schmalbruch H, Kato AC, Aebischer P (1995) Eur J Neurosci 7:1313–1322]. In contrast to CNTF, GDNF did not increase the lifespan of pmn/pmn mice. However, GDNF significantly reduced the loss of facial motoneurons by 50%, a value similar to what was observed when CNTF was administered to the pmn/pmn mice. Surprisingly, myelinated axon counts revealed that GDNF had no effect on nerve degeneration. Therefore, despite its potential in rescuing motoneuron cell bodies, the inability of GDNF to prevent nerve degeneration in pmn/pmn mice suggests that its usefulness in the treatment of motor neuron diseases may be restricted to cotreatment with other factors that act on the nerve process.

Keywords: Adenoviridae/genetics ; Animals ; Erythropoietin/ genetics/secretion ; Gene Therapy/ methods ; Genetic Vectors/administration a dosage ; Humans ; Models ; Animal ; Salivary Glands/ secretion ; Transduction ; Genetic/methods ; Transgenes ; Animal Note: The Institute of Neurosciences, Swiss Federal Institute of Technology Lausanne, EPFL. romain.zufferey@epfl.ch Reference LEN-ARTICLE-2004-011doi:10.1038/sj.gt.3302321View record in Web of Science Record created on 2007-03-09, modified on 2017-05-12

Reference LEN-ARTICLE-2004-015doi:10.1038/sj.gt.3302204View record in Web of Science Record created on 2008-08-27, modified on 2017-05-12

We previously demonstrated that Schwann cells (SCs) in semipermeable guidance channels promote axonal regeneration in adult rat spinal cord transected at the mid-thoracic level. Propriospinal but not supraspinal axons grew into these channels. Here, we tested the ability of exogenous brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) to promote axonal regeneration in this novel model. The two neurotrophins were delivered simultaneously into the channel by an Alzet minipump at a rate of 12 micrograms/day for each neurotrophin for 14 of 30 days tested; phosphate-buffered saline, the vehicle solution, was used as a control. Significantly more myelinated nerve fibers were present in SC/neurotrophin grafts than in SC/vehicle grafts (1523 +/- 292 vs 882 +/- 287). In the graft, at least 5 mm from the rostral cord-graft interface, some nerve fibers were immunoreactive for serotonin, a neurotransmitter specific to raphe-derived axons in rat spinal cord. Fast blue retrograde tracing from SC/neurotrophin grafts revealed labeled neurons in 10 nuclei of the brain stem, 67% of these being in the lateral and spinal vestibular nuclei. The mean number of labeled brain stem neurons in the SC/neurotrophin group (92; n = 3) contrasted with the mean in the SC/vehicle group (6; n = 4). Our results clearly demonstrate that BDNF and NT-3 infusion enhanced propriospinal axonal regeneration and, more significantly, promoted axonal regeneration of specific distant populations of brain stem neurons into grafts at the mid-thoracic level in adult rat spinal cord.

To attain light-dependent functionalization of biocompatible materials, a photolabel-derivatized, bioactive laminin fragment has been synthesized, chemically characterized, and photoimmobilized. Covalent high-resolution patterning of the laminin fragment CDPGYIGSR to hydroxylated fluorinated ethylene propylene (FEP-OH), poly(vinyl alcohol), and glycophase glass has been achieved. The synthetic peptide CDPGYIGSR was thermochemically coupled to either N-[m-[3-(trifluoromethyl)-diazirin-3-yl]phenyl]-4-maleimidobuty ramide or 4-maleimidobenzophenone. Photolabel-derivatized peptides were radiolabeled, and 20 and 300 microns-sized patterns were visualized by autoradiography. The biospecific interaction of photoimmobilized laminin fragments with cells was investigated by analyzing the selective attachment of NG 108-15 neuroblastoma x glioma cells which bear CDPGYIGSR-specific cell surface receptors. On photopatterned FEP-OH membranes NG 108-15 cells differentiated in serum-supplemented media within 1 day. Specific attachment to the immobilized oligopeptide CDPGYIGSR was assessed in serum-free media with competitive binding studies, showing an 82% decrease in cell adherence after the cell receptors were blocked with soluble CDPGYIGSR.

Ciliary neurotrophic factor (CNTF) has recently generated great interest due to its potential as a therapeutic agent for the treatment of human neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). Because the systemic half-life of CNTF is only in the order of a few minutes, continuous delivery of this trophic factor could be attractive or even necessary in the therapy of these diseases. One promising technique involves the polymer encapsulation of cells which have been genetically modified to secrete neurotrophic factors. The polymer capsules can be implanted into animals and effect the slow release of the protein for several months. The encapsulation technique immuno-isolates the foreign cells from host immune cells and at the same time prevents tumour formation by the transplanted cells. In this study, we have used progressive motoneuronopathy (pmn) mice to determine the extent to which encapsulated cell lines secreting CNTF could alter the course of the disease. pmn/pmn homozygotes present severe loss of myelinated motor fibres and a significant reduction of facial motoneuron cell bodies. The mice develop weakness of the hindlimbs and die during the sixth week after birth. We found that CNTF delayed the disease progression by increasing the survival time by 40% and by improving motor function as assessed by three behavioural tests. Moreover, histological counts of the phrenic nerve myelinated axons and facial nucleus motoneurons indicated a significant reduction of motoneuron loss. These results suggest that polymer-encapsulated cells releasing neurotrophic factors may provide a potential delivery system for treating neurodegenerative diseases such as ALS.

Material surfaces that can mediate cellular interactions by the coupling of specific cell membrane receptors may allow for the design of a biomaterial that can control cell attachment, differentiation, and tissue organization. Cell adhesion proteins have been shown to contain minimum oligopeptide sequences that are recognized by cell surface receptors and can be covalently immobilized on material surfaces. In this study, cell attachment to fluorinated ethylene propylene (FEP) films functionalized with the laminin-derived oligopeptides, YIGSR and a 19-mer IKVAV-containing sequence, was assessed using NG108-15 neuroblastoma and PC12 cells. A radiofrequency glow discharge (RFGD) process that replaces the FEP surface fluorine atoms with reactive hydroxyl functionalities was used to activate the film surfaces. The oligopeptides were then covalently coupled to the surface by their C-terminus using a standard nucleophilic substitution reaction. The covalent attachment of the oligopeptides to the FEP surface was verified using electron spectroscopy for chemical analysis (ESCA). Receptor-mediated NG108-15 cell attachment on the YIGSR-modified films was determined using competitive binding assays. Average cell attachment on the oligopeptide immobilized films in medium containing soluble CDPGYIGSR was reduced by approximately a factor of 2, compared to cell attachment in serum-free medium alone. No significant decrease in cell attachment was noted in medium containing the mock oligopeptide sequence CDPGYIGSK. FEP films immobilized with the 19-mer IKVAV sequence demonstrated a higher percentage of receptor mediated cell attachment on the film surfaces.(ABSTRACT TRUNCATED AT 250 WORDS)

Amyotrophic lateral sclerosis (ALS) is a progressive and fatal disease of the voluntary motor system. Its cause is unknown and no effective therapy has yet been devised. The natural history and range of expression of the disease is well known. It is a fatal disease, within 2 to 3 years of diagnosis. Although there is individual variation in the progression of the disease, quantitative measurement of the isometric force generated by a set of muscles allows one to follow the progression of the disease. This test, named the Tufts Quantitative Neuromuscular Exam (TQNE, Munsat et al., 1988), allows one to predict the individual patient’s course of disease on the basis of 3 successive tests, each one a month apart. The results of a novel therapy may therefore be assessed by looking for change in disease course versus the prediction.

We investigated the adsorption of albumin on chemically modified gold surfaces by scanning force microscopy operating both in contact and noncontact mode. The surface modification was performed with thiol-based self-assembling molecules carrying amine or methyl groups. The albumin on the aminoethanethiol-coated gold formed a uniform layer and single molecules could be distinguished. On the dodecanethiol-coated surface the protein adsorbed in aggregates or single isolated molecules depending on the incubation time. The width of the albumin molecule on both surface was similar, but the height was much lower on the amine than on the methyl surface. This was interpreted as a difference in the conformation of albumin depending on the substrate, and could explain the promotion of cell adhesion on amine-treated polymers coated with albumin.

Reference EPFL-ARTICLE-174091doi:10.1038/481260aView record in Web of Science Record created on 2012-01-19, modified on 2017-05-12

Glial cell line-derived neurotrophic factor (GDNF) has been postulated to be a specific dopaminergic neurotrophic factor since it selectively enhances the survival of dopaminergic neurones in vitro. We report here that GDNF can also act as a neurotrophic factor for motoneurones. GDNF released by GDNF-transfected BHK cells increases the activity of choline acetyltransferase (ChAT) in cultures from embryonic rat ventral mesencephalon containing cholinergic neurones from cranial motor nuclei and in cultured spinal motoneurones. Furthermore, local application of polymer-encapsulated BHK cells releasing GDNF to transected facial nerve in newborn rats diminishes the death of motoneurones normally occurring after axotomy in the neonatal period. The present results indicate that GDNF may have a therapeutic potential in human motoneurone diseases such as amyotrophic lateral sclerosis.

Gamma-aminobutyric acid (GABA)-releasing polymer matrices were implanted bilaterally, immediately dorsal to the substantia nigra, in rats previously kindled in the amygdala. Two days after implantation, rats with GABA-releasing matrices exhibited only focal limbic seizures in response to electrical stimulation, whereas animals with control matrices devoid of GABA had generalized convulsions. GABA release from the polymer matrices was high during the first days after implantation, as demonstrated both in vitro and, using microdialysis, in vivo. The anticonvulsant effect was no longer observed at 7 and 14 days at which time GABA release was found to be low. In a parallel experiment, polymer matrices containing noradrenaline (NA) were implanted bilaterally into the hippocampus of rats with extensive forebrain NA depletion induced by an intraventricular 6-hydroxydopamine injection. No effect on the development of hippocampal kindling was observed, despite extracellular NA levels exceeding those of rats with intrahippocampal locus coeruleus grafts that have previously been shown to retard kindling rate. The results indicate that GABA-releasing implants located in the substantia nigra region can suppress seizure generalization in epilepsy, even in the absence of synapse formation and integration with the host brain. In contrast, the failure of NA-releasing polymer matrices to retard the development of seizures in NA-depleted rats suggests that such an effect can only be exerted by grafts acting through a well-regulated, synaptic release of NA.

The patterned covalent surface addition of a monoamine to fluorinated ethylene propylene films (FEP) controls both cellular attachment and differentiation in defined media conditions. A radio frequency glow discharge (RFGD) process was used to replace FEP surface fluorine atoms with hydroxyl groups. The primary amine was then covalently attached by polymerizing aminopropyl-triethoxysilane (APTES) via the hydroxyl functionalities. The selective attachment of cells to the APTES regions was determined to be dependent upon the initial adsorption of albumin to the patterned FEP membrane. Albumin was determined to enhance cellular attachment to the APTES regions and prevent attachment to the unmodified FEP areas for both an NB2a neuroblastoma cell line and primary rat endothelial cells. If albumin were not preadsorbed onto the membrane, selective attachment to the modified regions would not occur. Radiolabeling albumin with 125I demonstrated the preference of albumin for adsorption onto the monoamine surface where the cells preferentially attached. Both hydrophobic and ionic forces contributed to the adsorption process. Although selective cellular attachment to the patterned APTES regions could be achieved by albumin preadsorption to the surface, the neuroblastoma cells did not significantly differentiate unless additional serum components were supplemented to the media.

Fluorinated ethylenepropylene copolymer (FEP) and polyvinylidene fluoride (PVDF) can generate static and transient electrical charges, respectively, after bulk molecular rearrangements induced by electrical charging techniques. Neurons cultured on electrically active FEP and PVDF show increased levels of nerve fiber outgrowth compared to electrically neutral material. The purpose of the present study was to determine if the addition of charged surface groups to the surfaces of FEP and PVDF would modify the influence of bulk electrical charges on cultured neurons. Mouse neuroblastoma (Nb2a) cells were cultured on electrically charged and uncharged FEP and PVDF substrates with covalently modified surfaces containing hydroxyl (OH) and amine (NH2) groups. Surface chemical modification was performed on the entire surface or in discrete striped regions. Nb2a cells cultured on electrically active FEP and PVDF showed greater levels of differentiation than cells on electrically neutral substrates. The presence of NH2 groups attenuated these responses in serum-containing media. Cells attached to NH2 rich surfaces generally displayed a flatter morphology and tended to remain attached for longer time periods. Cells cultured on stripe-modified substrates in serum-containing media showed a strong preferential attachment to modified regions, especially on NH2 stripes. In summary, bulk electrical charges are more important than surface charges in stimulating Nb2a cell differentiation. Surface groups serve to modulate neuronal morphology and confer specific attachment promoting properties in serum-containing media. The development of an optimal neuronal regeneration template may require the incorporation of specific bulk and surface properties.

A variety of neural electrodes have been developed to simulate and record from axons of the peripheral nervous system. Chronic neural interfaces may be used to facilitate voluntary movement or to control prosthetic devices in patients with spinal cord injuries, amputated appendages, or damaged nerves. Severed nerves are secured within synthetic tubular structures so as to induce and guide regenerating axons through neural interfaces. The regeneration process is strongly influenced by the properties of the tubular guidance channels. Channel characteristics impacting on regeneration include electrical, permeability, and surface microgeometrical properties as well as the ability to release growth factors. Biocompatible electrode materials are essential for insuring axonal survival and accurate signal transduction, especially in long-term use. By maximizing regeneration and providing a compatible neural interface, it may be possible to design implantable devices capable of chronic neural signal transduction in humans. Prosthetic devices and control units connected to neural interfaces could restore motor and sensory function. This review describes current approaches toward the development of nerve guidance channels and chronic neural interfaces.

Immobilization of discrete cell clusters within a partially crosslinked matrix prevents reaggregation of primary tissues and may provide a means for long-term maintenance of encapsulated cells. Dissociated bovine adrenal chromaffin (BAC) cells were suspended throughout crosslinked polyanionic microspheres previously shown to be selectively permeable. Microcapsules approximately 500 µm in diameter were seeded with: 1) three different densities of BAC cells; and 2) BAC cells suspended in Matrigel®or coseeded with a genetically modified nerve growth factor (NGF)- releasing fibroblast cell line. Each group was analyzedin vitroat 1, 4 and 8 weeks for spontaneous and potassium-evoked release of catecholamines, and maintainedin vitrofor up to 12 weeks for morphological observations. Over time, release of norepinephrine (NE) and epinephrine (EPI) diminished, while dopamine (DA) remained constant from the monoseeded capsules. In the coseeded group, an increase in potassium-evoked release of DA was observed from 1 to 4 weeks, and remained at that level up to 8 weeks. Encapsulated chromaffin cells retained a rounded morphology typical of undifferentiated cells. Intact chromaffin cells with well preserved and abundant secretory granules were observed ultrastructurally after 4 weeksin vitro. Small neurites from the chromaffin cells in the coseeded group were observed at 4 weeks with light microscopy, and up to 12 weeks with electron microscopy. Under static incubation conditions, 1 mM D-amphetamine resulted in a significant increase in the output of NE and DA from the coseeded capsules 8 weeks postimplantation, as compared to microcapsules loaded with chromaffin cells alone. Encapsulation within an immobilization matrix allows manipulation of the internal environment, thereby providing the ability to pre-treat cells with various factors in a non-invasive manner, which may enhance long-term cellular viability.

The use of conventional absorption based X-ray microtomography can become limited for samples showing only very weak absorption contrast. However, a wide range of samples studied in biology and materials science can produce significant phase shifts of the X-ray beam, and thus the use of the phase signal can provide substantially increased contrast and therefore new and otherwise inaccessible information. The application of two approaches for high-throughput, high-resolution X-ray phase contrast tomography, both available on the TOMCAT beamline of the SLS, is illustrated. Differential Phase Contrast (DPC) imaging uses a grating interferometer and a phase-stepping technique. It has been integrated into the beamline environment on TOMCAT in terms of the fast acquisition and reconstruction of data and the availability to scan samples within an aqueous environment. The second phase contrast approach is a modified transfer of intensity approach that can yield the 3D distribution of the phase (refractive index) of a weakly absorbing object from a single tomographic dataset. These methods are being used for the evaluation of cell integrity in 3D, with the specific aim of following and analyzing progressive cell degeneration to increase knowledge of the mechanistic events of neurodegenerative disorders such as Parkinson's disease.

Surrounding bovine chromaffin cells by a semipermeable membrane may protect the transplanted cells from a host immune response and shield them from the inflammatory process resulting from the surgical trauma. Encapsulation of the chromaffin cells was achieved by inter-facial adsorption of a polycation on a polyanionic colloid matrix in which the chromaffin cells were entrapped. Basal and potassium-evoked release of catecholamines from encapsulated bovine chromaffin cells was analyzed over a 4-week period in vitro. Norepinephrine and dopamine release remained constant over time whereas epinephrine release significantly decreased. The chromaffin cells also retained the capacity for depolarization-elicited catecholamine release 4 weeks following the encapsulation procedure. Morphological analysis revealed the presence of intact chromaffin cells with well-preserved secretory granules. Striatial implantation of chromaffin cell-loaded capsules significantly reduced apomorphine-induced rotation compared to empty polymer capsules in animals lesioned with 6-hydroxydopamne frr at least 4 weeks. Intact chromaffin cells expressing tyrosine hydroxylase and dopamine-β-hydroxylase were observed in all capsules implanted in the striatum for 4 weeks. The assessment of the clinical potential of transplanting encapsulated adrenal chromaffin cells of either allo- or xenogeneic origin for Parkinson's disease will require long-term behavioral studies. The present study suggests, however, that the polymer encapsulation procedure may offer an alternative to adrenal autografts as a source of dopaminergic tissue.

A new method of coextruding living cells in the core of a forming hollow fibre is described. PC12 cells, an immortalized cell line which secretes large amounts of dopamine, and dissociated bovine adrenal chromaffin cells, a non-dividing cell type which also secretes dopamine, were coextruded by a dry-jet wet spinning technique through a double-lumen spinneret from a 15% weight by volume solution of poly(acrylonitrile vinyl chloride) in either dimethylsulphoxide (DMSO), dimethylacetamide (DMAC) or dimethylformamide (DMF). Closure of the fibre was achieved by mounting polytetrafluoroethylene tubes on a rotating coaxial wheel system which squeezed the forming hollow fibre at regular intervals. Spontaneous and potassium-stimulated release of catecholamines from the macrocapsules were quantified under static conditions by ion-pair reverse-phase high-performance liquid chromatography equipped with electrochemical detection at 2, 4 and 6 wk. At all time periods, coextruded macrocapsules with either PC12 cells or adrenal chromaffin cells released dopamine under either unstimulated or stimulated conditions. An increase over time in dopamine release was observed from PC12 cell coextruded macrocapsules with observable difference between capsules extruded with DMSO, DMAC or DMF as solvents. Well-preserved PC12 cells and adrenal chromaffin cells were present in coextruded macrocapsules with no observable difference between capsules extruded with DMSO, DMAC or DMF as inocuity of macroencapsulation by coextrusion from an organic polymer solution. Owing to the particular fluid dynamics of this technique, minimal potentially toxic cell-solvent contact occurs allowing the use of a wider range of water-insoluble polymeric systems. The ability of encapsulated PC12 cells and adrenal chromaffin cells to release spontaneously dopamine over time also suggests that polymer encapsulation may provide an alternative to the transplantation of dopamine-secreting cells in the treatment of Parkinson's disease.

The present study was designed to evaluate the influence of synthetic guidance channel surface microgeometry on morphological patterns of neural regeneration. Tubes with smooth (S), rough (R), or alternating smooth-rough (S/R) or rough-smooth (R/S) inner surfaces but with identical chemical composition and permeability characteristics were used to bridge a 4-mm nerve gap in a transected mouse sciatic nerve. Animals received S and R channels for 1, 2 and 4 weeks and both S/R and R/S channels for 2 and 4 weeks. At 1 week, the S tubes contained a longitudinally oriented fibrin matrix not contacting the channel's smooth inner wall, whereas R tubes featured an unorganized fibrin matrix which, together with fibroblasts and macrophages, had invaded the channel's rough trabecular network. After 4 weeks, S tubes contained discrete, free-floating nerve cables with numerous myelinated and unmyelinated axons surrounded by a thin, continuous epineurial-like layer, whereas R tubes were completely filled with a loose connective tissue stroma with only a few axons. In combined S/R or R/S channels, the general morphological patterns in individual S or R segments were similar to those observed in pure S or R channels, regardless of whether the tube segment was positioned at the proximal or distal nerve end. Proximal smooth channel segments contained discrete cables which abruptly fanned out to completely fill the lumen in distal rough segments. The opposite pattern was observed with proximal rough and distal smooth segments. At 4 weeks, myelinated axons were observed along the entire length of S/R and R/S tubes. These results suggest that the surface microgeometry of guidance channels influences the outcome of peripheral nerve regeneration, potentially by affecting the early arrangement of the fibrin matrix and/or inducing different cellular responses.

Following a unilateral fimbria-fornix lesion, the delivery of nerve growth factor (NGF) to the ipsilateral lateral ventricle of the rat can prevent the lesion-induced loss of choline acetyltransferase (ChAT) expression in the ipsilateral medial septum and vertical diagonal band region. In the present study, the ability of polymer rods to deliver NGF and to prevent a decrease in basal forebrain ChAT expression following a fimbria-fornix lesion was assessed. NGF was loaded into an ethylene vinyl acetate copolymer (EVAc) rod, fabricated by a melt-extrusion process. NGF release was established by the ability of the rods to induce neurite extension from PC12 cells and chick E12 dorsal root ganglia. Unilateral aspirative lesions of the fimbria-fornix were performed in adult rats, followed by implantation of a polymer rod into the ipsilateral lateral ventricle. Five animals received EVAc rods containing only the carrier molecule bovine serum albumin (BSA), and six received EVAc rods containing both BSA and NGF. After 2 weeks, ChAT-positive cells were counted in the medial septum and vertical diagonal band regions. Rats with NGF-releasing rods displayed ChAT(+) cell counts ipsilateral to the lesion equal to 88% of those on the contralateral side. In contrast, ChAT(+) cell numbers were 42% in animals with rods releasing BSA only (P less than 0.001). No undue reaction to implanted rods was noted. Following a fimbria-fornix lesion, NGF released from polymer matrices effectively prevents a lesion-induced reduction in ChAT expression in basal forebrain neurons.

Our preliminary findings (Jaeger et al., 1988; Aebischer et al., 1989; Tresco et al., 1989) and the studies in progress show that encapsulated dopaminergic cell lines survive enclosure within a semi-permeable membrane. The encapsulated cells remained viable for extended time periods when maintained in vitro. Moreover, encapsulated PC12 and T28 cells have the potential to survive following their implantation into the forebrain of rats. Cell lines are essentially "immortal" because they continue to divide indefinitely. This property allows perpetual "self-renewal" of a given cell population. However, the capacity of continuous uncontrolled cell division may also lead to tumor formation. This in fact is the case for unencapsulated PC12 cell implants placed into the brain of young Sprague Dawley rats (Jaeger, 1985). Cell line encapsulation has the potential to prevent tumor growth (Jaeger et al., 1988). Survival for 6 months in vitro suggests that encapsulation does not preclude long-term maintenance of an homogeneous cell line like PC12 cells. The presence of mitotic figures in the capsules further supports the likelihood of propagation and self renewal of the encapsulated population. Another significant property of cell lines is that they consist of a single, genetically homogeneous cell type. They do not require specific synaptic interactions for their survival. In the case of PC12 and T28 lines, the cells synthesize and release neurotransmitters. Our data show that PC12 and T28 cells continue to release dopamine spontaneously and to express specific transmitters and enzymes following encapsulation. Thus, cell lines such as these may constitute relatively simple "neural implants" exerting their function via humoral release.(ABSTRACT TRUNCATED AT 250 WORDS)

Drug-inducible systems allowing the control of gene expression in mammalian cells are invaluable tools for genetic research, and could also fulfill essential roles in gene- and cell-based therapy. Currently available systems, however, often have limited in vivo functionality because of leakiness, insufficient levels of induction, lack of tissue specificity or prohibitively complicated designs. Here we describe a lentiviral vector-based, conditional gene expression system for drug-controllable expression of polymerase (Pol) II promoter-driven transgenes or Pol III promoter-controlled sequences encoding small inhibitory hairpin RNAs (shRNAs). This system has great robustness and versatility, governing tightly controlled gene expression in cell lines, in embryonic or hematopoietic stem cells, in human tumors xenotransplanted into nude mice, in the brain of rats injected intraparenchymally with the vector, and in transgenic mice generated by infection of fertilized oocytes. These results open up promising perspectives for basic or translational research and for the development of gene-based therapeutics.

Top of pageAbstract Lentiviral vectors expressing a mutant huntingtin protein (htt171-82Q) were used to generate a chronic model of Huntington's disease (HD) in rat primary striatal cultures. In this model, the majority of neurons expressed mutant huntingtin so that Western blot analysis and flow cytometry measurement could complement immunohistological evaluation. Mutant huntingtin produced a slowly progressing pathology characterized after 1 month by the appearance of neuritic aggregates followed by intranuclear inclusions, morphological anomalies of neurites, loss of neurofilament 160, increased expression in stress response protein HSP70 and later loss of neuronal markers such as NeuN and MAP-2. At 2 months post infection, a significant increase in TUNEL|[ndash]|positive cells confirmed actual striatal cell loss. Interestingly, cortical cultures infected with the same vector showed no sign of neuronal dysfunction despite accumulation of numerous inclusions. We finally examined whether, the trophic factors CNTF and BDNF which were found neuroprotective in acute HD models could prevent striatal degeneration in the present chronic model. Results demonstrated that both agents were neuroprotective without modifying inclusion formation. The present study demonstrates that using viral vectors coding for mutated Htt provides an advantageous system for histological and biochemical analysis of HD pathogenesis in primary striatal cultures.

Over 100 mutations in the Cu/Zn superoxide dismutase (SOD1) gene are linked to approximately 20% of familial ALS cases. Many strands of evidences suggest that SOD1 mutations cause motoneuron death by a gain of novel toxic property(ies). The therapeutic approach we propose consist in knocking down mutated SOD1, the primary element governing the whole pathogenic processes, via a viral-based delivery of small hairpin RNA specific to SOD1.

The CNS delivery of glial cell line-derived neurotrophic factor (GDNF) for the treatment of Parkinson's disease constitutes one of the more promising clinical applications of neurotrophic factors. Crucial for clinical application will be the ability to deliver GDNF within the target structures, i.e. striatum and/or substantia nigra. We are developing both in vivo and ex vivo gene therapy approaches to reach this goal. We have shown in rodents that both lentiviral vectors coding for GDNF and polymer encapsulated cells genetically engineered to release GDNF are able to protect nigral dopaminergic neurons against various insults including axotomy and neurotoxins such as 6-hydroxydopamine. Even more important for clinical application is the ability to scale-up the technology to nonhuman primate application. Neurorestorative and/or neuroprotective properties of GDNF expression were demonstrated with both methods in various nonhuman primate models.

Despite the success of current blood purification techniques in allowing the survival of individuals with acute and chronic renal failure, the quality of life of people affected by end-stage renal disease remains unsatisfactory. Part of the reason is due to the nonphysiologic manner in which current blood purification techniques achieve homeostasis. Attempts to improve mechanical substitution of renal function by coupling the transport capability of living cells with conventional hemofiltration devices constitute the first step toward the development of an implantable bioartificial kidney.

The brain tissue reaction to permselective polymer capsules implanted in rats was evaluated for 1 to 54 weeks. The polymer capsules were well tolerated in all animals and no recognizable neurological or behavioral deficits were associated with the implants. Necrosis at the brain/polymer interface, as assessed with Nissl stain, was not observed. Foreign body giant cells were consistently absent. Immunocytochemically identified reactive neuroglial cells showed a remarkably low-grade tissue response to the synthetic material be yond the first 2 weeks of observation. Immunolabeled cortical neurons revealed conserved columnar arrays around the implants. Transmission electron microscopy showed a minimal degree of collagen deposition compared to implants in peripheral sites, and normal synapses within a few micrometers from the brain/polymer interface, supporting the prospect of biocompatible, immunoisolated xenografts in the central nervous system.

Polytetrafluoroethylene (PTFE) tubes were prepared as electrets displaying a quasi-permanent surface charge due to the presence of trapped monopolar charge carriers. PTFE tubes containing either positive or negative charges and electrically neutral PTFE tubes were used as nerve guidance channels for the repair of a 4 mm nerve gap in the sciatic nerve of mice. After 4 weeks of implantation, positively and negatively charged PTFE electrets contained regenerated nerves with significantly more myelinated axons than nerves regenerated in uncharged PTFE tubes. This observation suggests that peripheral nerve regeneration can be enhanced by electrically charged nerve guidance channels.

The peritoneal cavity is a convenient site for implantation of encapsulated hormone-secreting tissue. However, host tissue organization around such implants may affect solute exchange and viability of the encapsulated tissue. The reaction to polyvinyl chloride acrylic copolymer capsules implanted in the peritoneal cavity of rats and mice was therefore studied. Some animals received a slow release dexamethasone pellet, others were pretreated with doxorubicin, in an attempt to minimize the tissue reaction. The tissue reaction was significantly thicker in rats than in mice at both 2 and 6 weeks after implantation. In rats, corticoids decreased significantly the thickness of the reactive layer as compared to control at all time points studied, but doxorubicin had no effect. The tissue reaction in mice was not significantly affected by corticoid treatment. In both species the thickness of the tissue reaction did not increase significantly between 2 and 6 weeks. At 3 days the tissue reaction consisted of an interrupted single layer of macrophages in mice, whereas in rats the reaction consisted of two or three layers of macrophages and polymorphonuclear cells. At both 2 and 6 weeks, several cell layers surrounded the implants: a single layer of macrophages lying along the polymer, a variable number of layers of fibroblasts interspersed with collagen fibrils (fewer in mice than in rats, and fewer in corticoid treated rats than control rats) and an outer monolayer of mesothelial cells. We conclude that the intensity of tissue reaction to polymer implants in the peritoneal cavity is species dependent and can be decreased by the administration of corticoids but not doxorubicin.