Eric Beaumont

Chitosan-DNA nanoparticles as non-viral vectors in gene therapy: strategies to improve transfection efficacy.

Currently, the major drawback of gene therapy is the gene transfection rate. The two main types of vectors that are used in gene therapy are based on viral or non-viral gene delivery systems. The viral gene delivery system shows a high transfection yield but it has many disadvantages, such as oncogenic effects and immunogenicity. However, cationic polymers, like chitosan, have potential for DNA complexation and may be useful as non-viral vectors for gene therapy applications. Chitosan is a natural non-toxic polysaccharide, it is biodegradable and biocompatible, and protects DNA against DNase degradation and leads to its condensation. The objective of this paper was to summarize the state of the art in gene therapy and particularly the use of chitosan to improve the transfection efficiency in vivo and in vitro.

Passive exercise and fetal spinal cord transplant both help to restore motoneuronal properties after spinal cord transection in rats

Spinal cord transection influences the properties of motoneurons and muscles below the lesion, but the effects of interventions that conserve muscle mass of the paralyzed limbs on these motoneuronal changes are unknown. We examined the electrophysiological properties of rat lumbar motoneurons following spinal cord transection, and the effects of two interventions shown previously to significantly attenuate the associated hindlimb muscle atrophy. Adult rats receiving a complete thoracic spinal cord transection (T‐10) were divided into three groups receiving: (1) no further treatment; (2) passive cycling exercise for 5 days/week; or (3) acute transplantation of fetal spinal cord tissue. Intracellular recording of motoneurons was carried out 4–5 weeks following transection. Transection led to a significant change in the rhythmic firing patterns of motoneurons in response to injected currents, as well as a decrease in the resting membrane potential and spike trigger level. Transplants of fetal tissue and cycling exercise each attenuated these changes, the latter having a stronger effect on maintenance of motoneuron properties, coinciding with the reported maintenance of structural and biochemical features of hindlimb muscles. The mechanisms by which these distinct treatments affect motoneuron properties remain to be uncovered, but these changes in motoneuron excitability are consistent with influences on ion conductances at or near the initial segment. The results may support a therapeutic role for passive limb manipulation and transplant of stem cells in slowing the deleterious responses of motoneurons to spinal cord injury, such that they remain more viable for subsequent alternative strategies. Muscle Nerve 29: 234–242, 2004

Effects of daily spontaneous running on the electrophysiological properties of hindlimb motoneurones in rats

No evidence currently exists that motoneurone adaptations in electrophysiological properties can result from changes in the chronic level of neuromuscular activity. We examined, in anaesthetized (ketamine/xylazine) rats, the properties of motoneurones with axons in the tibial nerve, from rats performing daily spontaneous running exercise for 12 weeks in exercise wheels (‘runners’) and from rats confined to plastic cages (‘controls’). Motoneurones innervating the hindlimb via the tibial nerve were impaled with sharp glass microelectrodes, and the properties of resting membrane potential, spike threshold, rheobase, input resistance, and the amplitude and time‐course of the afterhyperpolarization (AHP) were measured. AHP half‐decay time was used to separate motoneurones into ‘fast’ (AHP half‐decay time < 20 ms) and ‘slow’ (AHP half‐decay time ≥ 20 ms), the proportions of which were not significantly different between controls (58 % fast) and runners (65 % fast). Two‐way ANOVA and ANCOVA revealed differences between motoneurones of runners and controls which were confined to the ‘slow’ motoneurones. Specifically, runners had slow motoneurones with more negative resting membrane potentials and spike thresholds, larger rheobasic spike amplitudes, and larger amplitude AHPs compared to slow motoneurones of controls. These adaptations were not evident in comparing fast motoneurones from runners and controls. This is the first demonstration that physiological modifications in neuromuscular activity can influence basic motoneurone biophysical properties. The results suggest that adaptations occur in the density, localization, and/or modulation of ionic membrane channels that control these properties. These changes might help offset the depolarization of spike threshold that occurs during rhythmic firing.

Endurance training alters the biophysical properties of hindlimb motoneurons in rats

The purpose of the study was to determine the effect of daily endurance treadmill training (2 h/day, 30 m/min) on motoneuron biophysical properties. Electrophysiological properties of tibial motoneurons were measured in situ in anesthetized (ketamine/xylazine) control and trained rats using sharp glass microelectrodes. Motoneurons from trained rats had significantly hyperpolarized resting membrane potentials and spike trigger levels, and faster antidromic spike rise‐times. “Fast” motoneurons (after‐hyperpolarization half‐decay time <20 ms) in trained rats also had a significantly larger mean cell capacitance than those in control rats, suggesting that they were larger, although this had no effect on indices of excitability (rheobase, cell input resistance). Motoneurons are thus targets for activity‐induced adaptations, which may have clinical significance for the role of physical activity as a therapeutic modality in cases of neurological deficit. The specific adaptations noted, which reflect alterations in ionic conductances, may serve to offset decreases in membrane excitability that occur during sustained excitation.

Network interactions within the canine intrinsic cardiac nervous system: implications for reflex control of regional cardiac function

•  Control of regional cardiac function, as mediated by the intrinsic cardiac (IC) nervous system, is dependent upon its cardiac afferent neuronal inputs, changes in its central neuronal drive and interactions mediated within via local circuit neurons. •  The majority of its local circuit neurons receive indirect central (sympathetic and parasympathetic) inputs, lesser proportions transducing the cardiac milieu. •  Fifty per cent of IC neurons exhibit cardiac cycle‐related periodicity that is primarily related to direct cardiac mechano‐sensory afferent inputs and, secondarily, to indirect central autonomic efferent inputs. •  In response to mediastinal nerve stimulation, most IC neurons became excessively activated in the induction of atrial arrhythmias such that their stochastic interactivity precedes and persists throughout neuronally induced atrial fibrillation. •  Modulation of such stochastic IC local circuit neuronal recruitment may represent a novel target for the treatment of select cardiac disease, including atrial arrhythmias.

Hindlimb unweighting for 2 weeks alters physiological properties of rat hindlimb motoneurones

We sought to determine whether decreased neuromuscular use in the form of hindlimb unweighting (HU) would affect the properties of innervating motoneurones. Hindlimb weight‐bearing was removed in rats for a period of 2 weeks via hindlimb suspension by the tail. Following this the electrophysiological properties of tibial motoneurones were recorded under anaesthesia in situ. After HU, motoneurones had significantly (P < 0.05) elevated rheobase currents, lower antidromic spike amplitudes, lower afterhyperpolarization (AHP) amplitudes, faster membrane time constants, lower cell capacitances, and depolarized spike thresholds. Frequency–current (f–I) relationships were shifted significantly to the right (i.e. more current required to obtain a given firing frequency), although there was no change in f–I slopes. ‘Slow’ motoneurones (AHP half‐decay times, > 20 ms) were unchanged in proportions in HU compared to weight‐bearing rats. Slow motoneurones had significantly lower minimum firing frequencies and minimum currents necessary for rhythmic firing than ‘fast’ motoneurones in weight‐bearing rats; these differences were lost in HU rats, where slow motoneurones resembled fast motoneurones in these properties. In a five‐compartment motoneurone model with ion conductances incorporated to resemble firing behaviour in vivo, most of the changes in passive and rhythmic firing properties could be reproduced by reducing sodium conductance by 25% and 15% in the initial segment and soma, respectively, or by increasing potassium conductance by 55% and 42%, respectively. This supports previous conclusions that changes in chronic neuromuscular activity, either an increase or decrease, may result in physiological adaptations in motoneurones due to chronic changes in ion conductances.

Neuromodulation targets intrinsic cardiac neurons to attenuate neuronally mediated atrial arrhythmias

Our objective was to determine whether atrial fibrillation (AF) results from excessive activation of intrinsic cardiac neurons (ICNs) and, if so, whether select subpopulations of neurons therein represent therapeutic targets for suppression of this arrhythmogenic potential. Trains of five electrical stimuli (0.3-1.2 mA, 1 ms) were delivered during the atrial refractory period to mediastinal nerves (MSN) on the superior vena cava to evoke AF. Neuroanatomical studies were performed by injecting the neuronal tracer DiI into MSN sites that induced AF. Functional studies involved recording of neuronal activity in situ from the right atrial ganglionated plexus (RAGP) in response to MSN stimulation (MSNS) prior to and following neuromodulation involving either preemptive spinal cord stimulation (SCS; T(1)-T(3), 50 Hz, 200-ms duration) or ganglionic blockade (hexamethonium, 5 mg/kg). The tetramethylindocarbocyanine perchlorate (DiI) neuronal tracer labeled a subset (13.2%) of RAGP neurons, which also colocalized with cholinergic or adrenergic markers. A subset of DiI-labeled RAGP neurons were noncholinergic/nonadrenergic. MSNS evoked an ∼4-fold increase in RAGP neuronal activity from baseline, which SCS reduced by 43%. Hexamethonium blocked MSNS-evoked increases in neuronal activity. MSNS evoked AF in 78% of right-sided MSN sites, which SCS reduced to 33% and hexamethonium reduced to 7%. MSNS-induced bradycardia was maintained with SCS but was mitigated by hexamethonium. We conclude that MSNS activates subpopulations of intrinsic cardiac neurons, thereby resulting in the formation of atrial arrhythmias leading to atrial fibrillation. Stabilization of ICN local circuit neurons by SCS or the local circuit and autonomic efferent neurons with hexamethonium reduces the arrhythmogenic potential.

Neuroligin-1 links neuronal activity to sleep-wake regulation

Maintaining wakefulness is associated with a progressive increase in the need for sleep. This phenomenon has been linked to changes in synaptic function. The synaptic adhesion molecule Neuroligin-1 (NLG1) controls the activity and synaptic localization of N-methyl-d-aspartate receptors, which activity is impaired by prolonged wakefulness. We here highlight that this pathway may underlie both the adverse effects of sleep loss on cognition and the subsequent changes in cortical synchrony. We found that the expression of specific Nlg1 transcript variants is changed by sleep deprivation in three mouse strains. These observations were associated with strain-specific changes in synaptic NLG1 protein content. Importantly, we showed that Nlg1 knockout mice are not able to sustain wakefulness and spend more time in nonrapid eye movement sleep than wild-type mice. These changes occurred with modifications in waking quality as exemplified by low theta/alpha activity during wakefulness and poor preference for social novelty, as well as altered delta synchrony during sleep. Finally, we identified a transcriptional pathway that could underlie the sleep/wake-dependent changes in Nlg1 expression and that involves clock transcription factors. We thus suggest that NLG1 is an element that contributes to the coupling of neuronal activity to sleep/wake regulation.

Training improves the electrophysiological properties of lumbar neurons and locomotion after thoracic spinal cord injury in rats.

The aim of the present study was to evaluate the effect of a stepping-based rehabilitation program in voluntary wheel cages on the functional recovery and electrophysiological properties of neurons in the rat lumbar spinal cord after compressive thoracic (T10) spinal cord injury (SCI). A significant decrease in stance/swing duration and the number of limbs simultaneously in the stance phase was seen in trained compared to sedentary rats at 28 days after SCI (p<0.05). These kinematic improvements were associated with a significant increase in the amplitude of extracellular recordings from the tibial motoneuron pool in response to descending neuronal drive as well as significant amelioration of electrophysiological properties assessed from intracellular recordings. In fact, electrophysiological properties were not significantly different between uninjured controls and SCI-trained rats. Brain-derived neurotrophic factor (BDNF) levels were significantly elevated in the lumbar spinal cord of SCI-trained rats compared to SCI-sedentary controls. The data support a therapeutic role of increased neuromuscular activity in promoting functional recovery and suggest that it might occur via the beneficial effects of neurotrophic factors on neuronal plasticity.

Atorvastatin prevents early apoptosis after thoracic spinal cord contusion injury and promotes locomotion recovery.

The systemic administration of atorvastatin has been shown to be neuroprotective after spinal cord injury (SCI), by decreasing the inflammatory response at the lesion site and by reducing neuronal and oligodendrocyte apoptosis. The latter effect spares white matter at the injury site and improves locomotion. The aim of this study was to confirm the neuroprotective efficacy of atorvastatin as well as its early action in limiting apoptosis with its administration post-SCI. Female Sprague-Dawley rats received an intra peritoneal injection of: (1) statin/saline (5mg/kg) at 2h after the contusion injury; (2) physiological saline at 2h post-SCI; or (3) physiological saline without injury. Statin-treated rats showed significant (p<0.05) improvement in locomotion at week 4 post-SCI compared to vehicle-treated animals. Explaining this outcome, caspase-3 activity decreased by 50% (p<0.05), and the histological TUNEL method revealed a decrease of approximately 20% in apoptotic cells at the injury site (p<0.01) at 4h post-SCI in atorvastatin-treated rats in comparison to vehicle-treated controls. These data demonstrate that atorvastatin is effective after experimental spinal cord contusion injury in preventing early apoptosis at the injury site within 2h post-administration.