Romana Vavrek

Locomotion After Spinal Cord Injury Depends on Constitutive Activity in Serotonin Receptors

Following spinal cord injury (SCI) neurons caudal to the injury are capable of rhythmic locomotor-related activity that can form the basis for substantial functional recovery of stepping despite the loss of crucial brain stem-derived neuromodulators like serotonin (5-HT). Here we investigated the contribution of constitutive 5-HT(2) receptor activity (activity in the absence of 5-HT) to locomotion after SCI. We used a staggered hemisection injury model in rats to study this because these rats showed a robust recovery of locomotor function and yet a loss of most descending axons. Immunolabeling for 5-HT showed little remaining 5-HT below the injury, and locomotor ability was not correlated with the amount of residual 5-HT. Furthermore, blocking 5-HT(2) receptors with an intrathecal (IT) application of the neutral antagonist SB242084 did not affect locomotion (locomotor score and kinematics were unaffected), further indicating that residual 5-HT below the injury did not contribute to generation of locomotion. As a positive control, we found that the same application of SB242084 completely antagonized the muscle activity induced by exogenous application of the 5-HT(2) receptor agonists alpha-methyl-5-HT (IT). In contrast, blocking constitutive 5-HT(2) receptor activity with the potent inverse agonist SB206553 (IT) severely impaired stepping as assessed with kinematic recordings, eliminating most hindlimb weight support and overall reducing the locomotor score in both hind legs. However, even in the most severely impaired animals, rhythmic sweeping movements of the hindlimb feet were still visible during forelimb locomotion, suggesting that SB206553 did not completely eliminate locomotor drive to the motoneurons or motoneuron excitability. The same application of SB206553 had no affect on stepping in normal rats. Thus while normal rats can compensate for loss of 5-HT(2) receptor activity, after severe spinal cord injury rats require constitutive activity in these 5-HT(2) receptors to produce locomotion.

Transplantation and repair: Combined cell implantation and chondroitinase delivery prevents deterioration of bladder function in rats with complete spinal cord injury

Study design:Additional examination. In this study, we report changes in bladder function after a combined treatment that was designed to study axonal regeneration after complete spinal cord injury (SCI) in rats.Objectives:To report effects on bladder function following the administration of a combined treatment for complete SCI.Setting:University of Alberta, Faculty of Rehabilitation Medicine, Edmonton, CanadaMethods:Eight rats received Schwann cells in Matrigel-filled guidance channels, olfactory ensheathing glia and chondroitinase ABC at the lesion site following complete thoracic SCI. Controls (n=7) received Matrigel only. Daily bladder examinations were performed. Analysis of bladder size, wall thickness, actin and collagen type III was performed after 14 weeks.Results:Following SCI, both groups regained bladder voiding after 3 weeks. However, 2 weeks later, incontinence was observed in all untreated rats and two treated rats. Post-mortem examination of bladders revealed enlarged bladder sizes. Thicker bladder walls were found in untreated rats, which were composed of disorganized bundles of smooth muscle fibers surrounded by high amounts of collagen (type III).Conclusion:We show that the combined treatment prevents collagen deposition in bladder walls and maintains the rats ability to void efficiently. Although the mechanism responsible for this improvement is unclear, our study shows that the present combinatory therapy can influence bladder function, thus expanding their utility as a broad reparative approach for SCI.

Synthesis, transport, and metabolism of serotonin formed from exogenously applied 5-HTP after spinal cord injury in rats

Spinal cord transection leads to elimination of brain stem-derived monoamine fibers that normally synthesize most of the monoamines in the spinal cord, including serotonin (5-hydroxytryptamine, 5-HT) synthesized from tryptophan by enzymes tryptophan hydroxylase (TPH, synthesizing 5-hydroxytryptophan, 5-HTP) and aromatic l-amino acid decarboxylase (AADC, synthesizing 5-HT from 5-HTP). Here we examine whether spinal cord caudal to transection remains able to manufacture and metabolize 5-HT. Immunolabeling for AADC reveals that, while most AADC is confined to brain stem-derived monoamine fibers in spinal cords from normal rats, caudal to transection AADC is primarily found in blood vessel endothelial cells and pericytes as well as a novel group of neurons (NeuN positive and GFAP negative), all of which strongly upregulate AADC with injury. However, immunolabeling for 5-HT reveals that there is no detectable endogenous 5-HT synthesis in any structure in the spinal cord caudal to a chronic transection, including in AADC-containing vessels and neurons, consistent with a lack of TPH. In contrast, when we applied exogenous 5-HTP (in vitro or in vivo), AADC-containing vessels and neurons synthesized 5-HT, which contributed to increased motoneuron activity and muscle spasms (long-lasting reflexes, LLRs), by acting on 5-HT2 receptors (SB206553 sensitive) located on motoneurons (TTX resistant). Blocking monoamine oxidase (MAO) markedly increased the sensitivity of the motoneurons (LLR) to 5-HTP, more than it increased the sensitivity of motoneurons to 5-HT, suggesting that 5-HT synthesized from AADC is largely metabolized in AADC-containing neurons and vessels. In summary, after spinal cord injury AADC is upregulated in vessels, pericytes, and neurons but does not endogenously produce 5-HT, whereas when exogenous 5-HTP is provided AADC does produce functional amounts of 5-HT, some of which is able to escape metabolism by MAO, diffuse out of these AADC-containing cells, and ultimately act on 5-HT receptors on motoneurons.

Long-term viral brain-derived neurotrophic factor delivery promotes spasticity in rats with a cervical spinal cord hemisection.

We have recently reported that rats with complete thoracic spinal cord injury (SCI) that received a combinatorial treatment, including viral brain-derived neurotrophic factor (BDNF) delivery in the spinal cord, not only showed enhanced axonal regeneration, but also deterioration of hind-limb motor function. By demonstrating that BDNF over-expression can trigger spasticity-like symptoms in a rat model of sacral SCI, we proposed a causal relationship between the observed spasticity-like symptoms (i.e., resistance to passive range of motion) and the over-expression of BDNF. The current study was originally designed to evaluate a comparable combined treatment for cervical SCI in the rat to improve motor recovery. Once again we found similar signs of spasticity involving clenching of the paws and wrist flexion. This finding changed the focus of the study and, we then explored whether this spasticity-like symptom is directly related to the over-expression of BDNF by administering a BDNF antagonist. Using electromyographic measurements we showed that this treatment gradually diminished the resistance to overcome forelimb flexion in an acute experiment. Thus, we conclude that neuro-excitatory effects of chronic BDNF delivery together with diminished descending control after SCI can result in adverse effects.

Pericytes impair capillary blood flow and motor function after chronic spinal cord injury

Blood vessels in the central nervous system (CNS) are controlled by neuronal activity. For example, widespread vessel constriction (vessel tone) is induced by brainstem neurons that release the monoamines serotonin and noradrenaline, and local vessel dilation is induced by glutamatergic neuron activity. Here we examined how vessel tone adapts to the loss of neuron-derived monoamines after spinal cord injury (SCI) in rats. We find that, months after the imposition of SCI, the spinal cord below the site of injury is in a chronic state of hypoxia owing to paradoxical excess activity of monoamine receptors (5-HT1) on pericytes, despite the absence of monoamines. This monoamine-receptor activity causes pericytes to locally constrict capillaries, which reduces blood flow to ischemic levels. Receptor activation in the absence of monoamines results from the production of trace amines (such as tryptamine) by pericytes that ectopically express the enzyme aromatic L-amino acid decarboxylase (AADC), which synthesizes trace amines directly from dietary amino acids (such as tryptophan). Inhibition of monoamine receptors or of AADC, or even an increase in inhaled oxygen, produces substantial relief from hypoxia and improves motoneuron and locomotor function after SCI.

Dose and Chemical Modification Considerations for Continuous Cyclic AMP Analog Delivery to the Injured CNS

In this investigation, two cell-permeable synthetic analogs of cAMP, dibutyryl-cAMP (db-cAMP) and 8-bromo-cAMP, which are widely used to elevate intracellular cAMP levels under experimental conditions, were investigated for their ability to dose-dependently improve histological and functional outcomes following continuous delivery in two models of incomplete spinal cord injury (SCI). The cAMP analogs were delivered via osmotic minipumps at 1-250 mM through an indwelling cortical cannula or by intrathecal infusion for up to 4 weeks after either a T8 unilateral over-hemisection or a C2-3 dorsolateral quadrant lesion, respectively. In both SCI models, continuous db-cAMP delivery was associated with histopathological changes that included sporadic micro-hemorrhage formation and cavitation, enhanced macrophage infiltration and tissue damage at regions beyond the immediate application site; no deleterious or beneficial effect of agent delivery was observed at the spinal injury site. Furthermore, these changes were accompanied by pronounced behavioral deficits that included an absence of progressive locomotor recovery, increased extensor tone, paralysis, and sensory abnormalities. These deleterious effects were not observed in saline-treated animals, in animals in which the db-cAMP dose did not exceed 1 mM, or in those animals that received a high dose (250 mM) of the alternative cAMP analog, 8-bromo-cAMP. These results demonstrate that, for continuous intraparenchymal or intrathecal administration of cAMP analogs for the study of biological or therapeutic effects within the central nervous system (CNS), consideration of the effective concentration applied as well as the potential toxicity of chemical moieties on the parent molecule and/or their activity needs to be taken into account.

Training following unilateral cervical spinal cord injury in rats affects the contralesional forelimb

Rehabilitative training is an essential component of current therapeutic strategies for spinal cord injured individuals. However, there are still various open questions that need to be answered in order to optimize training strategies. For example, why can animals trained in a single task perform worse compared to untrained animals when tested in untrained tasks. Such results suggest a potential competition among motor tasks over spared neuronal circuitry. Whether training induced competition for neuronal circuitry may also exist between injured and spared circuitries of the ipsi- and contralesional extremity is currently unknown. Here we investigated whether training restricted to the frontlimb ipsilateral to cervical spinal injury (IF) can impact motor performance of the contralesional frontlimb (CF) in a rat model of cervical SCI. We compared CF performance following general motor training of all limbs (horizontal ladder), following specific training of the IF (pellet reaching), as well as following a combination of both training paradigms. Our findings indicate that adding ipsilateral side-specific training to general training can negatively impact performance of the CF, without resulting in any improvement of performance of the IF. In conclusion, our results emphasize that important decisions have to be made when designing rehabilitative training strategies, ideally taking into account more than the primarily affected extremity.

Eliciting inflammation enables successful rehabilitative training in chronic spinal cord injury

Rehabilitative training is one of the most successful therapies to promote motor recovery after spinal cord injury, especially when applied early after injury. Polytrauma and management of other medical complications in the acute post-injury setting often preclude or complicate early rehabilitation. Therefore, interventions that reopen a window of opportunity for effective motor training after chronic injury would have significant therapeutic value. Here, we tested whether this could be achieved in rats with chronic (8 weeks) dorsolateral quadrant sections of the cervical spinal cord (C4) by inducing mild neuroinflammation. We found that systemic injection of a low dose of lipopolysaccharide improved the efficacy of rehabilitative training on forelimb function, as assessed using a single pellet reaching and grasping task. This enhanced recovery was found to be dependent on the training intensity, where a high-intensity paradigm induced the biggest improvements. Importantly, in contrast to training alone, the combination of systemic lipopolysaccharide and high-intensity training restored original function (reparative plasticity) rather than enhancing new motor strategies (compensatory plasticity). Accordingly, electrophysiological and tract-tracing studies demonstrated a recovery in the cortical drive to the affected forelimb muscles and a restructuration of the corticospinal innervation of the cervical spinal cord. Thus, we propose that techniques that can elicit mild neuroinflammation may be used to enhance the efficacy of rehabilitative training after chronic spinal cord injury.