Acute intermittent hypoxia enhances regeneration of surgically repaired peripheral nerves in a manner akin to electrical stimulation.

The intrinsic repair response of injured peripheral neurons is enhanced by brief electrical stimulation (ES) at time of surgical repair, resulting in improved regeneration in rodents and humans. However, ES is invasive. Acute intermittent hypoxia (AIH) - breathing alternate cycles of regular air and air with ~50% normal oxygen levels (11% O2), considered mild hypoxia, is an emerging, promising non-invasive therapy that promotes motor function in spinal cord injured rats and humans. AIH can increase neural activity and under moderately severe hypoxic conditions improves repair of peripherally crushed nerves in mice. Thus, we posited an AIH paradigm similar to that used clinically for spinal cord injury, will improve surgically repaired peripheral nerves akin to ES, including an impact on regeneration-associated gene (RAG) expression-a predictor of growth states. Alterations in early RAG expression were examined in adult male Lewis rats that underwent tibial nerve coaptation repair with either 2 days AIH or normoxia control treatment begun on day 2 post-repair, or 1 h ES treatment (20 Hz) at time of repair. Three days post-repair, AIH or ES treatments effected significant and parallel elevated RAG expression relative to normoxia control at the level of injured sensory and motor neuron cell bodies and proximal axon front. These parallel impacts on RAG expression were coupled with significant improvements in later indices of regeneration, namely enhanced myelination and increased numbers of newly myelinated fibers detected 20 mm distal to the tibial nerve repair site or sensory and motor neurons retrogradely labeled 28 mm distal to the repair site, both at 25 days post nerve repair; and improved return of toe spread function 5-10 weeks post-repair. Collectively, AIH mirrors many beneficial effects of ES on peripheral nerve repair outcomes. This highlights its potential for clinical translation as a non-invasive means to effect improved regeneration of injured peripheral nerves.

Behavioural analysis of the efficacy of treatments for injuries to the spinal cord in animals.

There have been very few clinical trials evaluating therapies for naturally occurring spinal cord injury in dogs and cats. This review describes the methods suitable for evaluating the behavioural recovery of animals with spinal cord injuries, in either a clinical or a laboratory setting. A list of commonly used methods for evaluating behavioural recovery in animals is provided, both in the clinical and laboratory setting; the tests, their limitations and benefits and specific recommendations for their use are also discussed in more depth.

Posthatching locomotor experience alters locomotor development in chicks.

We have previously demonstrated that, even though chicks are very precocial and can locomote within hours of hatching, they require a period of time to develop a mature stable walk. As an example, 1- to 2-day-old animals move with disproportionately small stride lengths compared with 10- to 14-day-old animals. The purpose of this study was to determine whether the maturation of walking, including the development of a mature stride length, depends on locomotor experience. We also investigated the development and experience-dependence nature of head bobbing, an optokinetic behavior that occurs during walking in birds. Chicks were randomly assigned to one of three groups receiving either increased locomotor experience (i.e., treadmill exercise), decreased locomotor experience (i.e., decreased housing space), or no alteration in locomotor experience. To assess the dependence of locomotor maturation on N-methyl-D-aspartate (NMDA)-type glutamate receptors, animals in each group were either given an NMDA antagonist (MK-801, 1 mg/kg intramuscularly daily) or saline control. Locomotor characteristics (stride length, leg support durations, horizontal head excursions) were quantified from videotaped recordings of chicks walking overground unrestrained on posthatching days 1, 2, 4, 6, 8, and 10. Animals subject to exercise restriction for at least 6 days moved with shortened stride lengths compared with age-matched treadmill-exercised or control animals, a change that was maintained for the duration of the study. NMDA antagonism also resulted in shortened stride lengths. Head bobbing behavior matured during the same posthatching time period. The rate of this maturation was also decreased by exercise restriction. Thus locomotor experience is required for normal development of locomotor behavior, even in very precocial animals. These results are discussed in terms of the possible neuroanatomical and neurophysiological mechanisms underlying experience- and activity-dependent changes during motor development.

Mini-review: assessment of behavioural recovery following spinal cord injury in rats.

Behavioural recovery is one of the primary goals of therapeutic intervention in animal models of disease. It is necessary, therefore, to have the means with which to quantify pertinent behavioural changes in experimental animals. Nevertheless, the number and diversity of behavioural measures which have been used to assess recovery after experimental interventions often makes it difficult to compare results between studies. The present review attempts to integrate and categorize the wide variety of behavioural assessments used to measure recovery in spinal-injured rats. These categories include endpoint measures, kinematic measures, kinetic measurements, and electrophysiological measurements. Within this categorization, we discuss the advantages and disadvantages of each type of measurement. Finally, we make some recommendations regarding the principles for a comprehensive behavioural analysis after experimental spinal cord injury in rats.

The blood-brain barrier and its role in inflammation.

The unique microenvironment within the central nervous system (CNS) relies upon the integrity of the blood-brain barrier (BBB). This selectively permeable barrier comprises interendothelial tight junctions located at the capillaries and postcapillary venules. Cells and structures in the local environment are required to maintain normal BBB function. When inflammation is present, the BBB itself plays an integral role in the inflammatory response by either producing or expressing a variety of cytokines, adhesion molecules, metalloproteinases, serine proteases, products of arachidonic acid metabolism, and nitric oxide. Understanding the role of the BBB during inflammation is essential when creating and employing a therapeutic regime for animals with CNS disease. This review focusses on recent discoveries about the BBB and its role in inflammation, and applies this knowledge to our current understanding of inflammatory CNS disease in dogs and cats.

Red nucleus lesions impair overground locomotion in rats: a kinetic analysis.

The red nucleus is a prominent brainstem nucleus in mammals which is thought to be involved in production of skilled limb movements. The presence of the red nucleus and associated rubrospinal tract in animals that do not produce skilled limb movements, however, suggests that these structures might also be involved in control of more general limb actions, such as those occurring during locomotion. The present study investigates this question by measuring the three-dimensional ground reaction forces produced by locomoting rats with unilateral excitotoxic lesions of the red nucleus. Twenty-four to 48 h after the lesion, rats moved with an asymmetric gait during which abnormal braking and propulsive forces were produced during the dual contact time of the forelimb contralateral to the lesion and the ipsilateral hindlimb. Rats did not recover normal symmetrical locomotion within the 55-day duration of the study. The persistent asymmetry produced by red nucleus ablation provides the first unequivocal demonstration that the red nucleus plays a role in ongoing overground locomotion in the rat. Species differences in phylogeny and connectivity of the red nucleus are discussed, as well as the possibility that there is a general compensatory response to unilateral CNS injury in the rat.

Early ontogeny of locomotor behaviour: a comparison between altricial and precocial animals.

The focus of this review is to examine the physiological and behavioural differences between the early ontogeny of locomotion in precocial and altricial species. Both groups of animals are capable of performing alternating stepping movements upon birth or hatching, indicating that the basic elements underlying locomotor synergy are present prior to expression of mature overground gait. Nevertheless, the notable difference between precocial and altricial animals is the ability of the former to walk and run soon after birth or hatching. The weight of experimental evidence suggests that postural constraints play an important role in preventing early expression of locomotor behaviour in altricial species. Even some precocial animals, however, need time to develop sufficient stability and balance to walk as an adult. Therefore, components of locomotor behaviour involving the maintenance of equilibrium need a period of maturation in both precocial and altricial species, possibly requiring locomotor experience to become fully mature.

Locomotor plasticity after spinal injury in the chick.

Functional recovery after spinal cord injury likely depends, in part, on the reorganization of undamaged spinal circuitry. Segmental afferent input from the limbs remains largely intact after spinal injury and may provide an important source of activation and regulation of the spinal circuits that have lost descending input as a result of the injury. This purpose of this study was to investigate the contribution of cutaneous afferent inputs to the recovery of motor function after spinal injury in the chick. After lateral thoracic spinal hemisection, the motion of the ipsilateral limb was impaired during both walking and swimming. By 2 weeks postoperatively, limb motion recovered to preoperative values for walking but not for swimming. It was hypothesized that phasic afferent inputs experienced during walking, but not swimming, contributed to recovery of limb motion during walking. When a source of phasic cutaneous input was provided during swim training sessions, limb motion gradually improved to preoperative values. After 2 weeks of training, this improved motion was retained even after the source of cutaneous stimulation was removed. The proposed mechanism is an experience-dependent strengthening of the circuits activated during the improved limb motion, leading to a permanent change in limb action during swimming. Thus, the afferent inputs experienced during movement repetition are important during the acquisition of learned movements after spinal injury. These results are discussed in terms of behavioral, physiological, and anatomical evidence for spinal plasticity in other species. It is concluded that the spinal cord has significant plastic capabilities, and efforts should be directed toward maximizing the contribution of this plasticity to functional recovery after spinal cord injury.

Complete locomotor recovery following corticospinal tract lesions: measurement of ground reaction forces during overground locomotion in rats.

The corticospinal tract is a prominent descending pathway in rodents which is thought to be involved in motor control. The purpose of this study was to investigate whether the lesions of the corticospinal tract affected overground locomotion in rats using a method of assessment which is relatively novel in this field, the evaluation of ground reaction forces. Ground reaction forces, i.e. the forces acting through the limbs on the ground, are a non-invasive, sensitive and quantitative method with which to assess gait compensation and the function of individual limbs during locomotion. We compared the three-dimensional ground reaction forces produced by locomoting rats with a unilateral corticospinal tract transection to those of control rats. Corticospinal-lesioned animals showed transient locomotor deficits 24-48 h after the lesion which quickly recovered to normal symmetrical locomotion. The initial locomotor deficits consisted of an asymmetric gait during which abnormal braking forces were produced during the dual contact time of the forelimb contralateral to the lesion (the impaired forelimb) and the ipsilateral (unimpaired) hindlimb. Normal forces were produced during the dual contact time of the ipsilateral (unimpaired) forelimb and the contralateral (impaired) hindlimb. The presence of the initial deficits may be a result of inflammation in the area of the lesion or may reflect a loss of normal locomotor contribution of the corticospinal tract. If the latter, the nature of the locomotor deficit suggests that (a) the forelimb may be more influenced by corticospinal lesions than is the hindlimb and/or (b) that the asymmetric gait produced is a general compensatory response to unilateral CNS injury in a quadruped. Complete recovery from corticospinal tract transection provides unequivocal evidence that input from the corticospinal tract is not essential for normal overground locomotion in the rat.

Ground reaction forces in locomoting hemi-parkinsonian rats: a definitive test for impairments and compensations.

Hemi-parkinsonian rats have preserved postural reflexes but are impaired in initiation of voluntary movements. Surprisingly, these rats can walk and run, suggesting that they can access some compensatory strategy to overcome the rigidity in their impaired limbs. The purpose of the present experiment was to investigate the locomotor compensations made by hemi-parkinsonian rats by measuring the forces exerted by the limbs on the ground throughout the stride during trotting. Rats with unilateral dopamine depletion produced by injection of 6-hydroxydopamine into the nigrostriatal bundle were trained to run back and forth in an alley for food reinforcement. Ground reaction forces were measured in three orthogonal directions using a force plate embedded in the runway. Rats were also videotaped so that limb movements were synchronized with force recordings. Although locomotion was obviously impaired, the affected limbs could support weight and provide some braking forces. In addition, the impaired hindlimb provided significant propulsive force, and a relatively large laterally directed force. Analysis of vertical movement of the centre of mass suggested that the impaired hindlimb was being used partly as a spring. The most significant abnormalities were seen during the diagonal couplet of the impaired forelimb and the unimpaired hindlimb, partly reflecting the important compensatory role of the unimpaired hindlimb. These results demonstrate that this method is useful in the analysis of hemi-parkinsonian gait and provide insights as to how rats can use an impaired limb to produce weight support and propulsion.

Asymmetric bipedal locomotion--an adaptive response to incomplete spinal injury in the chick.

The purpose of this study was to compare the asymmetric gait induced by unilateral spinal cord injury in chicks with asymmetric gaits of other bipeds and quadrupeds. After lateral hemisection of the left thoracic spinal cord, kinetic (ground reaction forces) and kinematic (distance and timing) data were recorded as chicks moved overground unrestrained. Ground reaction forces were analyzed to obtain the mechanical energy changes throughout the stride. Kinematic measurements were obtained over a range of speeds to determine the velocity-dependent characteristics of the gait. Hemisected chicks adopted an asymmetric hopping gait in which the animals hopped from the right leg (contralateral to the lesion) onto the left (ipsilateral) leg but then fell forward onto the right leg. Mechanical energy fluctuations throughout a single stride (i.e., two steps) approximated the oscillations that occur during a single walking step of control animals. When examined over a range of velocities, asymmetries in limb timing remained constant, but distance measurements such as step length became more symmetric as speed increased. The results show that, after spinal hemisection, adaptations of the remaining neural circuitry permitted the production of a locomotor pattern that, in addition to providing effective support and propulsion, incorporated some of the energy-conserving mechanisms of the normal walk. Adjustment of this novel locomotor pattern for different velocities further demonstrates the flexibility of locomotor circuitry. Comparisons with other studies shows that this gait shares some temporal and energetic features with asymmetric gaits of several bipedal species, including humans. In particular, hemisected chicks and some hemiplegic humans adopt an asymmetric gait in which maximum energy recovery occurs during the stance of the affected limb; these similarities probably relate to common mechanical constraints imposed on bipedal forms of terrestrial locomotion.

Sensorimotor stimulation to improve locomotor recovery after spinal cord injury.

Functional recovery after CNS injury may depend, in part, upon reorganization of undamaged neural pathways. Spinal cord circuits are capable of significant reorganization, in the form of both activity-dependent and injury-induced plasticity. This plasticity is manifest behaviourally in the ability of spinal animals to learn new locomotor tasks. Recent work with spinal-injured humans demonstrates that training can improve functional locomotor abilities. New methodologies to enhance limb movement are designed to exploit further the plastic capabilities of the spinal cord by reinforcing appropriate connections in an activity-dependent manner. In the future, these methods might also prove useful in guiding and strengthening functional synaptogenesis of regenerating axons to maximize their contribution towards restoration of function.

Ontogeny of bipedal locomotion: walking and running in the chick.

1. The purpose of this study was to determine whether the production of an energy-efficient bipedal walk is an innate attribute of a precocial bird. 2. The locomotor characteristics of hatchling chicks were quantified using kinetic (ground reaction forces) and kinematic (stride length, leg support duration) measurements as the animals moved overground unrestrained. All measurements were made over a range of velocities and at regular intervals throughout the first 2 weeks of life. 3. Ground reaction force records showed that, like all terrestrial walking vertebrates, chicks undergo cyclical increases and decreases in the body's potential and kinetic energy with each step. The out-of-phase exchange of potential with kinetic energy is an efficient mechanism for the conservation of energy during walking. However, comparisons between chicks at posthatching (P) days 1-2 and P14 revealed that P1-2 chicks are unable to conserve energy because they walk with disproportionately small potential energy oscillations. During running, however, the oscillations between potential and kinetic energy are similar for both P1-2 and P14 animals. 4. P1-2 chicks also walk with a shorter stride length than P14 chicks. Examination of limb support durations shows that younger animals (P1-2, P3) spend less time in single limb support than P14 animals during walking but not running. 5. The results show that even highly precocial bipeds need to acquire the ability to walk in a controlled and energy efficient manner, although they can innately run as well as an adult. This disparity could be due to the distinct actions of the legs in these two behaviours, and the requirement for longer durations of single leg support during walking. These differences relate to constraints inherent to bipedal locomotion and many of the locomotor changes occurring in the first weeks after hatching may therefore be analogous to similar changes seen during human locomotor development.

Phasic cutaneous input facilitates locomotor recovery after incomplete spinal injury in the chick.

1. Walking and swimming of hatchling chicks was videotaped before hemisection of the left thoracic cord and thereafter at regular intervals, for up to 2 wk. With the use of kinematic techniques, movements of the left knee and ankle were quantified to assess recovery of the ipsilateral leg during walking and swimming trials. To study the effects of exteroceptive (cutaneous) feedback in the absence of limb loading, one group of animals was also provided with cutaneous stimulation during swimming in the form of neutrally buoyant tubes that only contacted the foot during the retraction (extension) phase of the swim cycle. 2. One day after hemisection, for both swimming and walking, the left knee failed to extend normally, and the ankle joint remained hyperextended. During walking, all chicks adopted an asymmetric gait, whereas during swimming the left leg remained retracted and motionless. 3. Over the next 2 wk, knee extension and ankle flexion during walking recovered to normal preoperative values, but neither measure returned to preoperative values during swimming trials. However, when chicks were provided with phasic cutaneous stimulation during swimming trials, they showed improvements in leg motion as soon as 5 days after hemisection. Temporary removal of the cutaneous stimulation during swimming (5 days after hemisection) resulted in reduced limb action. However, 14 days after hemisection, the improvement in limb motion was retained even when the cutaneous stimulation was not provided. 4. Improvement in leg motion after swim training with phasic cutaneous stimulation took the form of increased extension of the limb during retraction. Possible neurophysiological mechanisms for this behavior include reflex reinforcement of limb extensor activity in response to cutaneous stimulation of the foot. Repeated exposure to phasic stimulation during swimming trials results in a permanent alteration in limb action. Thus increased cutaneous afferent inputs, even in the absence of limb loading, can facilitate locomotor recovery after spinal cord injury.

Axonal regeneration contributes to repair of injured brainstem-spinal neurons in embryonic chick.

Recent results have demonstrated complete anatomical and functional repair of descending brainstem-spinal projections in chicken embryos that underwent thoracic spinal cord transection prior to embryonic day 13 (E13) of the 21 d developmental period. To determine to what extent axonal regeneration was contributing to this repair process, we conducted experiments using a double retrograde tract-tracing protocol. On E8-E13, the upper lumbar spinal cord was injected with the first fluorescent tracing dye to label those brainstem-spinal neurons projecting to the lumbar cord at that time. One to two days later (on E10-E15), the upper to mid-thoracic spinal cord was completely transected. After an additional 7-8 d, a different second fluorescent tracing dye was injected into the lumbar cord at least 5 mm caudal to the site of transection. Finally, 2 d later on E19 to postnatal day 4, the CNS was fixed and sectioned. Brainstem and spinal cord tissue sections were then viewed with epifluorescence microscopy. In comparison to nontrasected control animals, our findings indicated that there were relatively normal numbers of double-labeled brainstem-spinal neurons after a transection prior to E13, whereas the number of double-labeled and second-labeled brainstem-spinal neurons decreases after an E13-E15 transection. In addition, at each subsequent stage of development from E10 to E12, there was a greater number of double-labeled brainstem-spinal neurons (indicating regeneration of previously severed axons) than cell bodies labeled with the second fluorescent tracer alone (indicating subsequent development of late brainstem-spinal projections). Assessment of voluntary open-field locomotion (hatchling chicks) and/or brainstem-evoked locomotion (embryonic or hatchling) indicated that functional recovery of animals transected prior to E13 was indistinguishable from that observed in control chicks (sham operated or unoperated). Taken together, these data suggest that regeneration of previously axotomized fibers contributes to the observed anatomical and functional recovery after an embryonic spinal cord transection.

Suppression of the onset of myelination extends the permissive period for the functional repair of embryonic spinal cord.

In an embryonic chicken, transection of the thoracic spinal cord prior to embryonic day (E) 13 (of the 21-day developmental period) results in complete neuroanatomical repair and functional locomotor recovery. Conversely, repair rapidly diminishes following a transection on E13-E14 and is nonexistent after an E15 transection. The myelination of fiber tracts within the spinal cord also begins on E13, coincident with the transition from permissive to restrictive repair periods. The onset of myelination can be delayed (dysmyelination) until later in development by the direct injection into the thoracic cord on E9-E12 of a monoclonal antibody to galactocerebroside, plus homologous complement. In such a dysmyelinated embryo, a subsequent transection of the thoracic cord as late as E15 resulted in complete neuroanatomical repair and functional recovery (i.e., extended the permissive period for repair).

Odor of the muskox : A preliminary investigation.

The behavior of captive male muskoxen was observed closely during their characteristic superiority display, the anatomy of the preputial region was studied in two adults and three calves, and preputial washings and preorbital gland secretion were subjected to gas chromatography and mass spectroscopy. During the superiority display, the prepuce was everted to form a pendulous tube tipped with a fringe of matted hair. Owing to the movement of the animal, the urine that dribbled from the preputial opening was liberally applied to the long guard hairs of the belly. The superiority display was almost exclusively confined to dominant males and apparently accounted for their odor. In the quiescent state, the hair seen around the preputial opening was drawn inside and formed an 8 cm-wide band on the lining of the prepuce. The preputial washings contained large amounts of benzoic acid andp-cresol. The infraorbital gland secretion contained cholesterol, benzaldehyde, and a homologous series of saturated γ-lactones ranging from 8 to 12 carbons. The latter compounds and the natural secretion smell similar to the human nose.