Neutralization of CD95 ligand promotes regeneration and functional recovery after spinal cord injury

The clinical outcome of spinal cord injury (SCI) depends in part on the extent of secondary damage, to which apoptosis contributes. The CD95 and tumor necrosis factor (TNF) ligand/receptor systems play an essential role in various apoptotic mechanisms. To determine the involvement of these ligands in SCI-induced damage, we neutralized the activity of CD95 ligand (CD95L) and/or TNF in spinal cord-injured mice. Therapeutic neutralization of CD95L, but not of TNF, significantly decreased apoptotic cell death after SCI. Mice treated with CD95L-specific antibodies were capable of initiating active hind-limb movements several weeks after injury. The improvement in locomotor performance was mirrored by an increase in regenerating fibers and upregulation of growth-associated protein-43 (GAP-43). Thus, neutralization of CD95L promoted axonal regeneration and functional improvement in injured adult animals. This therapeutic strategy may constitute a potent future treatment for human spinal injury.     

Neurotrophic factors and their receptors in axonal regeneration and functional recovery after peripheral nerve injury

Over a half a century of research has confirmed that neurotrophic factors promote the survival and process outgrowth of isolated neurons in vitro. The mechanisms by which neurotrophic factors mediate these survival-promoting effects have also been well characterized. In vivo, peripheral neurons are critically dependent on limited amounts of neurotrophic factors during development. After peripheral nerve injury, the adult mammalian peripheral nervous system responds by making neurotrophic factors once again available, either by autocrine or paracrine sources. Three families of neurotrophic factors were compared, the neurotrophins, the GDNF family of neurotrophic factors, and the neuropoetic cytokines. Following a general overview of the mechanisms by which these neurotrophic factors mediate their effects, we reviewed the temporal pattern of expression of the neurotrophic factors and their receptors by axotomized motoneurons as well as in the distal nerve stump after peripheral nerve injury. We discussed recent experiments from our lab and others which have examined the role of neurotrophic factors in peripheral nerve injury. Although our understanding of the mechanisms by which neurotrophic factors mediate their effects in vivo are poorly understood, evidence is beginning to emerge that similar phenomena observed in vitro also apply to nerve regeneration in vivo.     

Rapid differentiation of superficial urothelial cells after chitosan-induced desquamation

Superficial cell desquamation followed by differentiation of newly exposed superficial cells induces regeneration of the urinary bladder epithelium, urothelium. In the present work, chitosan was evaluated as a new inducer of urothelial cell desquamation, in order to study the regeneration of mouse urothelial cells in vivo. Intravesical application of chitosan dispersion caused complete removal of only the superficial layer of cells within 20 min of treatment. Differentiation of the new superficial layer was followed by the appearance and distribution of three urothelial differentiation markers, tight junction protein ZO1, cytokeratin 20 and the maturation of the apical plasma membrane. The arrangement of ZO1 into continuous lines in individual cells of the intermediate layer was already found after 10 min of chitosan application, when desquamation had just started. The appearance of the apical membrane changed from microvillar to typically scalloped within 20 min of regeneration, while complete arrangement of the cytokeratin 20 network took 60 min. These findings provide a new perspective on the rate of the differentiation process in the urothelium and make chitosan a new and a very controllable tool for studies on urothelial regeneration.     

Early functional muscle regeneration after myotoxic injury in mice is unaffected by nNOS absence

Nitric oxide (NO) is an important signaling molecule produced in skeletal muscle primarily via the neuronal subtype of NO synthase (NOS1, or nNOS). While many studies have reported NO production to be important in muscle regeneration, none have examined the contribution of nNOS-derived NO to functional muscle regeneration (i.e., restoration of the muscle's ability to produce force) after acute myotoxic injury. In the present study, we tested the hypothesis that genetic deletion of nNOS would impair functional muscle regeneration after myotoxic injury in nNOS(-/-) mice. We found that nNOS(-/-) mice had lower body mass, lower muscle mass, and smaller myofiber cross-sectional area and that their tibialis anterior (TA) muscles produced lower absolute tetanic forces than those of wild-type littermate controls but that normalized or specific force was identical between the strains. In addition, muscles from nNOS(-/-) mice were more resistant to fatigue than those of wild-type littermates (P < 0.05). To determine whether deletion of nNOS affected muscle regeneration, TA muscles from nNOS(-/-) mice and wild-type littermates were injected with the myotoxin notexin to cause complete fiber degeneration, and muscle structure and function were assessed at 7 and 10 days postinjury. Myofiber cross-sectional area was lower in regenerating nNOS(-/-) mice than wild-type controls at 7 and 10 days postinjury; however, contrary to our original hypothesis, no difference in force-producing capacity of the TA muscle was evident between the two groups at either time point. Our findings reveal that nNOS is not essential for functional muscle regeneration after acute myotoxic damage.     

A fine structural study of regeneration after fission in the planarian Dugesia japonica

We examined morphologically the process of regeneration before and after fission in a sexual strain of the freshwater planarian Dugesia japonica. Usually fission takes place in the post-pharyngeal region. Decapitation significantly accelerates the rate of fissioning. When decapitated worms were treated with substance P and neuropeptide K separately, the rate of fission markedly decreased in both cases. Before the onset of fission, a presumptive region of fission was recognized in the post-pharyngeal portion where undifferentiated cells, regenerative cells and newly differentiated cells were localized. Moreover a functional network of fixed parenchyma cells was noted in this region. After fission, cell distribution in the blastema became quite different from that of artificially amputated worms. This difference seems to be due to the process that occurs in the presumptive region of fission. This revised version was published online in July 2006 with corrections to the Cover Date.     

Lentiviral-mediated transfer of CDNF promotes nerve regeneration and functional recovery after sciatic nerve injury in adult rats

Peripheral nerve injury is often followed by incomplete and unsatisfactory functional recovery and may be associated with sensory and motor impairment of the affected limb. Therefore, a novel method is needed to improve the speed of recovery and the final functional outcome after peripheral nerve injuries. This report investigates the effect of lentiviral-mediated transfer of conserved dopamine neurotrophic factor (CDNF) on regeneration of the rat peripheral nerve in a transection model in vivo. We observed notable overexpression of CDNF protein in the distal sciatic nerve after recombinant CDNF lentiviral vector application. We evaluated sciatic nerve regeneration after surgery using light and electron microscopy and the functional recovery using the sciatic functional index and target muscle weight. HE staining revealed better ordered structured in the CDNF-treated group at 8 weeks post-surgery. Quantitative analysis of immunohistochemistry of NF200 and S-100 in the CDNF group revealed significant improvement of axonal and Schwann cell regeneration compared with the control groups at 4 weeks and 8 weeks after injury. The thickness of the myelination around the axons in the CDNF group was significantly higher than in the control groups at 8 weeks post-surgery. The CDNF group displayed higher muscle weights and significantly increased sciatic nerve index values. Our findings suggest that CDNF gene therapy could provide durable and stable CDNF protein concentration and has the potential to enhance peripheral nerve regeneration, morphological and functional recovery following nerve injury, which suggests a promising strategy for peripheral nerve repair.     

Diphenylpiperazines enhance regeneration after facial nerve injury

Immature rat facial motoneurons are very sensitive to injury with nearly 80% dying during the first week after axotomy. This motoneuron death is apoptotic, similar to that induced in neurons after tropic factor withdrawal. The diphenylpiperazines, flunarizine and cinnarizine, protect dorsal root ganglion neurons from death after withdrawal of trophic support, i.e., nerve growth factor withdrawal, in vitro. Similarly, the monoamine oxidase inhibitor, deprenyl, promotes survival of facial motoneurons after axotomy. These pharmacological agents were assessed both alone and in combination for their ability to prevent death in non-nerve growth factor dependent CNS motoneurons after facial nerve axotomy in newborn rats. Long-term experiments were done with the diphenylpiperazines to evaluate potential enhancement of regeneration. Facial nerve transection resulted in 78% neuronal loss in the injured compared with the contralateral, uninjured nucleus. Systemic administration of diphenylpiperazines for 1 week after facial nerve transection doubled the number of surviving motoneurons from 23% to 47%. Similar results were obtained with deprenyl. Combinations of diphenylpiperazines and deprenyl provide a similar degree of neuronal protection 1 week after injury as that obtained by either agent alone. We assessed the ability of diphenylpiperazines to protect facial motoneurons from death over a prolonged period and enhance subsequent regeneration. Motor neuron counts in rats treated with diphenylpiperazines for 1 month after injury and assessed 2 months later demonstrated long-term enhancement of neuronal protection with an increase of 45% in the number of horseradish peroxidase-labelled motoneurons. The diphenylpiperazines group had ～80% more regenerated myelinated axons in the distal facial nerve than the control group. Thus, diphenylpiperazine treatment during the first month after injury provides long-term protection of non-nerve growth factor dependent CNS motoneurons with subsequent potentiation of long-term facial nerve regeneration.     

Botulinum toxin A inhibits ATP release from bladder urothelium after chronic spinal cord injury

The effects of mechanoreceptor stimulation and subsequent ATP release in spinal cord injured and normal bladders was examined to demonstrate if spinal cord injury (SCI) modulates the basal or evoked release of ATP from bladder urothelium and whether intravesical botulinum toxin A (BTX-A) administration inhibits urothelial ATP release, a measure of sensory nerve activation. A Ussing chamber was used to isolate and separately measure resting and mechanoreceptor evoked (e.g. hypoosmotic stimulation) ATP release from urothelial and serosal sides of the bladder. Following spinal cord injury, resting urothelial release of ATP was ninefold higher than that of normal rats. Botulinum toxin A instillation did not significantly affect the resting release of ATP after spinal cord injury. Evoked ATP release following hypoosmotic stimulation was significantly higher in chronic spinal cord injured compared to normal rat bladders. However, botulinum toxin A treatment markedly reduced ATP release in spinal cord injured bladders by 53% suggesting that ATP release by mechanoreceptor stimulation, as opposed to basal release, occurs by exocytotic mechanisms. In contrast, there was no significant difference in basal or evoked ATP release from bladder serosa following spinal cord injury. Moreover, intravesical instillation of botulinum toxin A did not affect ATP release from the serosal side after spinal cord injury, suggesting that its effects were confined to the urothelial side of the bladder preparation. In summary: (1) increased release of ATP from the urothelium of spinal cord injured bladders may contribute to the development of bladder hyperactivity and, (2) mechanoreceptor stimulated vesicular ATP release, as opposed to basal non-vesicular release of ATP, is significantly inhibited in spinal cord injured bladders by intravesical instillation of botulinum toxin A. These results may have important relevance in our understanding of the mechanisms underlying plasticity of bladder afferent pathways following SCI.     

Lymphatic vessels in bone support regeneration after injury

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Intramuscular beta2-agonist administration enhances early regeneration and functional repair in rat skeletal muscle after myotoxic injury.

Systemic administration of beta(2)-adrenoceptor agonists (beta(2)-agonists) can improve skeletal muscle regeneration after injury. However, therapeutic application of beta(2)-agonists for muscle injury has been limited by detrimental cardiovascular side effects. Intramuscular administration may obviate some of these side effects. To test this hypothesis, the right extensor digitorum longus (EDL) muscle from rats was injected with bupivacaine hydrochloride to cause complete muscle fiber degeneration. Five days after injury, half of the injured muscles received an intramuscular injection of formoterol (100 mug). Muscle function was assessed at 7, 10, and 14 days after injury. A single intramuscular injection of formoterol increased muscle mass and force-producing capacity at day 7 by 17 and 91%, respectively, but this effect was transient because these values were not different from control levels at day 10. A second intramuscular injection of formoterol at day 7 prolonged the increase in muscle mass and force-producing capacity. Importantly, single or multiple intramuscular injections of formoterol did not elicit cardiac hypertrophy. To characterize any potential cardiovascular effects of intramuscular formoterol administration, we instrumented a separate group of rats with indwelling radio telemeters. Following an intramuscular injection of formoterol, heart rate increased by 18%, whereas systolic and diastolic blood pressure decreased by 31 and 44%, respectively. These results indicate that intramuscular injection can enhance functional muscle recovery after injury without causing cardiac hypertrophy. Therefore, if the transient cardiovascular effects associated with intramuscular formoterol administration can be minimized, this form of treatment may have significant therapeutic potential for muscle-wasting conditions.     

Neurotrophic factor gene delivery supports axonal regeneration after injury

A successful treatment for spinal cord injury (SCI) must include means to induce axonal regeneration and synaptogenesis. Though much research has demonstrated the effectiveness of neurotrophic factors (NFs) in supporting axonal regeneration, systemic delivery of doses sufficient to reach therapeutic concentrations and overcome their short half-lives has caused adverse effects. Local expression of NFs would overcome these limitations. We tested whether local expression of NFs would induce axonal regeneration without adverse effects in two models of neural injury. In a chemical injury model the rat serotonergic system was lesioned with p-chloroamphetamine. When an adenoviral vector carrying the gene for brain-derived neurotrophic factor (BDNF) was injected into the denervated cortex BDNF expressed by the transfected cells induced serotonergic axon reinnervation only in area around the injection site. In a mechanical injury model the cortical spinal tract (CST) in rats was lesioned unilaterally at the level of the hindbrain. Neurotorphin-3 (NT-3) was expressed locally in the spinal cord either by direct injection of an adenoviral vector carrying the gene for NT-3 or by retrograde delivery of the vector from the sciatic nerve. Axons were observed growing from the unlesioned CST across the midline to the denervated side. These data demonstrate that local expression of NFs will induce and support axonal regeneration in a circumscribed area after injury without adverse effects and suggest that a therapy for SCI based upon this strategy may include NF gene delivery.
Acknowledgements:   Supported by NIH grant NS35280 and Mission Connect of the TIRR Foundation.     

Mouse gastrocnemius muscle regeneration after mechanical or cardiotoxin injury

The goal of our study was to compare the skeletal muscle regeneration induced by two types of injury: either crushing, that causes muscle degeneration as a result of mechanical devastation of myofibers, or the injection of a cardiotoxin that is a myotoxic agent causing myolysis of myofibers leading to muscle degeneration. Regenerating muscles were analyzed at selected intervals, until the 14th day following the injury. We analyzed their weight and morphology. We also studied the expression of different myosin heavy chain isoforms as a molecular marker of the regeneration progress. Histological analysis revealed that inflammatory response and myotube formation in crushed muscles was delayed compared to cardiotoxin-injected ones. Moreover, the expression of myosin heavy chain isoforms was observed earlier in cardiotoxin-injured versus crushed muscles. We conclude that the dynamics of skeletal muscle regeneration depends on the method of injury.     

Neurotrophin treatment to promote regeneration after traumatic CNS injury

Neurotrophins are a family of growth factors that have been found to be central for the development and functional maintenance of the nervous system, participating in neurogenesis, neuronal survival, axonal growth, synaptogenesis and activity-dependent forms of synaptic plasticity. Trauma in the adult nervous system can disrupt the functional circuitry of neurons and result in severe functional deficits. The limitation of intrinsic growth capacity of adult nervous system and the presence of an inhospitable environment are the major hurdles for axonal regeneration of lesioned adult neurons. Neurotrophic factors have been shown to be excellent candidates in mediating neuronal repair and establishing functional circuitry via activating several growth signaling mechanisms including neuron-intrinsic regenerative programs. Here, we will review the effects of various neurotrophins in mediating recovery after injury to the adult spinal cord.     

Neurotrophic factor gene delivery supports axonal regeneration after injury

A successful treatment for spinal cord injury (SCI) must include means to induce axonal regeneration and synaptogenesis. Though much research has demonstrated the effectiveness of neurotrophic factors (NFs) in supporting axonal regeneration, systemic delivery of doses sufficient to reach therapeutic concentrations and overcome their short half‐lives has caused adverse effects. Local expression of NFs would overcome these limitations. We tested whether local expression of NFs would induce axonal regeneration without adverse effects in two models of neural injury. In a chemical injury model the rat serotonergic system was lesioned with p‐chloroamphetamine. When an adenoviral vector carrying the gene for brain‐derived neurotrophic factor (BDNF) was injected into the denervated cortex BDNF expressed by the transfected cells induced serotonergic axon reinnervation only in area around the injection site. In a mechanical injury model the cortical spinal tract (CST) in rats was lesioned unilaterally at the level of the hindbrain. Neurotorphin‐3 (NT‐3) was expressed locally in the spinal cord either by direct injection of an adenoviral vector carrying the gene for NT‐3 or by retrograde delivery of the vector from the sciatic nerve. Axons were observed growing from the unlesioned CST across the midline to the denervated side. These data demonstrate that local expression of NFs will induce and support axonal regeneration in a circumscribed area after injury without adverse effects and suggest that a therapy for SCI based upon this strategy may include NF gene delivery. Acknowledgements: Supported by NIH grant NS35280 and Mission Connect of the TIRR Foundation.     

Posture shifting after spinal cord injury using functional neuromuscular stimulation--a computer simulation study

The ability for individuals with spinal cord injury (SCI) to affect changes in standing posture with functional neuromuscular stimulation (FNS) was explored using an anatomically inspired musculoskeletal model of the trunk, pelvis and lower extremities (LE). The model tracked trajectories for anteriorly and laterally shifting movements away from erect stance. Forces were applied to both shoulders to represent upper extremity (UE) interaction with an assistive device (e.g., a walker). The muscle excitations required to execute shifting maneuvers with UE forces <10% body-weight (BW) were determined via dynamic optimization. Nine muscle sets were examined to maximize control of shifting posture. Inclusion of the Psoas and External Obliques bilaterally resulted in the least relative UE effort (0.119, mean UE effort = 45.3N ≡ 5.4% BW) for anterior shifting. For lateral shifting, the set including the Psoas and Latissimus Dorsi bilaterally yielded the best performance (0.025, mean UE effort = 27.8 N ≡ 3.3% BW). However, adding the Psoas alone bilaterally competed favorably in overall best performance across both maneuvers. This study suggests suitable activation to specific muscles of the trunk and LE can enable individuals with SCI to alter their standing postures with minimal upper-body effort and subsequently increase reach and standing work volume.     

Integrins promote axonal regeneration after injury of the nervous system

Integrins are cell surface receptors that form the link between extracellular matrix molecules of the cell environment and internal cell signalling and the cytoskeleton. They are involved in several processes, e.g. adhesion and migration during development and repair. This review focuses on the role of integrins in axonal regeneration. Integrins participate in spontaneous axonal regeneration in the peripheral nervous system through binding to various ligands that either inhibit or enhance their activation and signalling. Integrin biology is more complex in the central nervous system. Integrins receptors are transported into growing axons during development, but selective polarised transport of integrins limits the regenerative response in adult neurons. Manipulation of integrins and related molecules to control their activation state and localisation within axons is a promising route towards stimulating effective regeneration in the central nervous system.     

Molecular, cellular and developmental biology of urothelium as a basis of bladder regeneration

Urinary bladder malfunction and disorders are caused by congenital diseases, trauma, inflammation, radiation, and nerve injuries. Loss of normal bladder function results in urinary tract infection, incontinence, renal failure, and end-stage renal dysfunction. In severe cases, bladder augmentation is required using segments of the gastrointestinal tract. However, use of gastrointestinal mucosa can result in complications such as electrolyte imbalance, stone formation, urinary tract infection, mucous production, and malignancy. Recent tissue engineering techniques use acellular grafts, cultured cells combined with biodegradable scaffolds, and cell sheets. These techniques are not all currently applicable for human bladder reconstruction. However, new avenues for bladder reconstruction maybe facilitated by a better understanding of urogenital development, the cellular and molecular biology of urothelium, and cell-cell interactions, which modulate tissue repair, homeostasis, and disease progression.