Transplants of fibroblasts genetically modified to express BDNF promote regeneration of adult rat rubrospinal axons and recovery of forelimb function.
(17/1162)
Adult mammalian CNS neurons do not normally regenerate their severed axons. This failure has been attributed to scar tissue and inhibitory molecules at the injury site that block the regenerating axons, a lack of trophic support for the axotomized neurons, and intrinsic neuronal changes that follow axotomy, including cell atrophy and death. We studied whether transplants of fibroblasts genetically engineered to produce brain-derived neurotrophic factor (BDNF) would promote rubrospinal tract (RST) regeneration in adult rats. Primary fibroblasts were modified by retroviral-mediated transfer of a DNA construct encoding the human BDNF gene, an internal ribosomal entry site, and a fusion gene of lacZ and neomycin resistance genes. The modified fibroblasts produce biologically active BDNF in vitro. These cells were grafted into a partial cervical hemisection cavity that completely interrupted one RST. One and two months after lesion and transplantation, RST regeneration was demonstrated with retrograde and anterograde tracing techniques. Retrograde tracing with fluorogold showed that approximately 7% of RST neurons regenerated axons at least three to four segments caudal to the transplants. Anterograde tracing with biotinylated dextran amine revealed that the RST axons regenerated through and around the transplants, grew for long distances within white matter caudal to the transplant, and terminated in spinal cord gray matter regions that are the normal targets of RST axons. Transplants of unmodified primary fibroblasts or Gelfoam alone did not elicit regeneration. Behavioral tests demonstrated that recipients of BDNF-producing fibroblasts showed significant recovery of forelimb usage, which was abolished by a second lesion that transected the regenerated axons. (+info)
Material properties and biosynthetic activity of articular cartilage from the bovine carpo-metacarpal joint.
(18/1162)
OBJECTIVE: To determine the site variation of material properties and cellular biosynthetic activity, and to compare these at each site, of articular cartilage from the bovine carpo-metacarpal joint in order to test its usefulness as a model system for investigating the metabolic effects of mechanical stimuli. DESIGN: The mechanical properties and composition of full-depth biopsies of articular cartilage were measured at defined sites in bovine carpometacarpal joints. Metabolic activity at the same sites was assessed by incubating further biopsies in medium containing 35S-sulfate and 3H-leucine then measuring the incorporated radioisotope and cell density. These results were compared with an estimate of the distribution of forces across the joint. RESULTS: Topographical variation of water content, stiffness, cell count or incorporated radioisotope was not significant whereas collagen and glycosaminoglycan varied in different ways. A moderate correlation was found between water and collagen contents, but the correlation between water and glycosaminoglycan contents was poor. Neither compressive stiffness nor creep compliance was predicted strongly by any of the composition measurements. A negative correlation was found between metabolic activity and cell density. CONCLUSIONS: Defining the variation of tissue properties across the bovine carpometacarpal joint and the lack of variation in biosynthetic activity will enable proper matching of experimental and control groups of biopsies in studies of the effects of mechanical stimuli on the composition and mechanical properties of articular cartilage. In addition, the lack of correlation between stiffness, water and glycosaminoglycan contents is further evidence that the mechanical properties of the tissue depend significantly on factors other than these broad measures of composition. (+info)
Direct exposure of postimplantation mouse embryos to 5-bromodeoxyuridine in vitro and its effect on subsequent chondrogenesis in the limbs.
(19/1162)
As maternally administered 5-bromodeoxyuridine (BudR) is very quickly degraded by the liver, a combination of whole embryo culture and organ culture techniques was adopted to expose postimplantation mouse embryos to the analog and to study the effects of long-term embryos were exposed to increasing concentrations of BudR for 12 or 24 h. Forelimbs of the treated embryos were then organ-cultured in drug-free medium and the extent of cartilage development in the explants examined. Exposure of embryos to 50-150 mug/ml of BudR for 24 h resulted in significant inhibition of chondrogenesis in the subsequent limb cultures and the effect was related to dose. After treatment with 150 mug/ml of the drug, the forelimbs of the early 11-day embryos (somite stage 26-29) showed an almost complete lack of cartilage, while the limbs of mid-11 th-day embryos (somite stage 32-34) were not nearly as sensitive and exhibited about 50% reduction in the amount of cartilage development. We conclude that if embryos in which the limb development is at a very early stage of development are exposed to BudR, the future course of limb differentiation is permanently and irreversibly damaged, resulting in a partial or even complete suppression of chondrogenesis in the organ. As both the dose and perhaps also the duration of treatment were critical, we suggest that the rather low frequency of reported limb malformations after in vivo injection of teratological doses of BudR may be due to only a small amount of the chemical reaching the embryos. (+info)
Positionally selective growth of embryonic spinal cord neurites on muscle membranes.
(20/1162)
Motor neurons from distinct positions along the rostrocaudal axis generally innervate muscles or muscle fibers from corresponding axial levels. These topographic maps of connectivity are partially restored after denervation or transplantation under conditions in which factors of timing and proximity are eliminated. It is therefore likely that motor neurons and some intramuscular structures bear cues that bias synapse formation in favor of positionally matched partners. To localize these cues, we studied outgrowth of neurites from embryonic spinal cord explants on carpets of membranes isolated from perinatal rat muscles. Neurites from rostral (cervical) and caudal (lumbar) spinal cord slices exhibit distinct growth preferences. In many instances, rostrally derived neurites grew selectively on membranes from forelimb muscles or from a single thoracic muscle (the serratus anterior) when given a choice between these membranes and membranes from hindlimb muscles or laminin. Caudally derived neurites almost never exhibited such rostral preferences, but instead preferred membranes from hindlimb muscles or a single hindlimb muscle (the gluteus) to rostral muscles or laminin. Likewise, spinal neurites exhibited distinct position-related preferences for outgrowth on membranes of clonal myogenic cell lines derived from specific rostral and caudal muscles. Taken together these results suggest that the membranes of motor axons and myotubes bear complementary labels that vary with rostrocaudal position and regulate neuromuscular connectivity. (+info)
Monosynaptic Ia pathways at the cat shoulder.
(21/1162)
1. The study aimed to describe in cat forelimb and shoulder motoneurones the convergence and projection patterns from large muscle spindle afferents (Ia). In 11 chloralose-anaesthetized cats maximum Ia EPSPs evoked by electrical stimulation of ipsilateral forelimb nerves were obtained in 309 intracellularly recorded alpha-motoneurones. 2. Groups of motor nuclei displayed similar Ia patterns. As in the distal forelimb they were often interconnected by bidirectional pathways, which were used to combine Ia synergistic groups. Three such groups are described at the shoulder. 3. The first group was composed of the main flexors of the scapulo-humeral joint. Regular disto-proximal Ia excitation from elbow extensors (and median afferents) indicates a coupling of flexion in the scapulo-humeral joint to the angular position of the elbow. 4. The second group comprised the outward rotators of the humerus with differentiated Ia convergence onto the different group members. The patterns of Ia excitation received and sent by the group members demonstrate that the outward rotators are incorporated in versatile synergisms and may occupy a central position in steering forelimb movements. 5. The third group was formed by the spinatus muscle and the subscapularis. This arrangement is suggested by the common convergence onto them from the elbow extensors and flexors. The pattern may serve to guide and keep the humeral head in the joint capsule. 6. The Ia synergistic groups receive Ia convergence from muscles acting at distant joints and also project to distant muscles. This is discussed as part of an extended pattern of Ia connections along the forelimb. In this way the shoulder muscles would be incorporated in flexor and extensor oriented synergisms which are needed to co-ordinate the muscular activation along the multijoint forelimb during locomotion. When the shoulder Ia pathways are compared with those in the distal forelimb, organization of the Ia system apparently follows a few basic principles which have adapted to the mechanical situation at the particular joints and their mechanical interaction. (+info)
Central regulation of motor cortex neuronal responses to forelimb nerve inputs during precision walking in the cat.
(22/1162)
1. The responses of neurones in forelimb motor cortex to impulse volleys evoked by single pulse electrical stimulation (at 1.5 or 2 times the threshold for most excitable nerve fibres) of the superficial radial (SR) and ulnar (UL) nerves of the contralateral forelimb were studied in awake cats both resting quietly and walking on a horizontal ladder. Nerve volley amplitude was monitored by recording the compound action potential elicited by the stimulus. 2. In the resting animal 34/82 (41%) cells yielded statistically significant responses to SR stimulation, and 20/72 (28%) responded to UL stimulation. Some responses were confined to or began with an increase in firing probability ('excitatory' responses) and others with a decrease in firing ('inhibitory' responses), typically including a brief interruption of the spike train (zero rate). Cells responding to both nerves usually yielded responses similar in type. Most (78%) response onset latencies were less than 30 ms. Responses involved the addition or subtraction of from 3.4 to 0.1 impulses stimulus-1 (most <1 impulse stimulus-1). The distribution of response sizes was continuous down to the smallest values, i.e. there was no 'gap' which would represent a clear separation into 'responsive' and 'unresponsive' categories. Responses were commonest in the lateral part of the pericruciate cortex, and commoner among pyramidal tract neurones (PTNs) than non-PTNs. 3. During ladder walking most cells generated a rhythmic step-related discharge; in assessing the size of responses to nerve stimulation (20 studied, from 13 cells) this activity was first subtracted. Response onset latencies (90% <30 ms) and durations showed little or no change. Although most cells were overall more active than during rest both 'excitatory' and 'inhibitory' responses in both PTNs and non-PTNs were often markedly reduced in large parts of the step cycle; over some (usually brief) parts responses approached or exceeded their size during rest, i.e. response size was step phase dependent. Such variations occurred without parallel change in the nerve compound action potential, nor were they correlated with the level of background firing at the time that the response was evoked. When responses to both nerves were studied in the same neurone they differed in their patterns of phase dependence. 4. The findings are interpreted as evidence for central mechanisms that, during 'skilled', cortically controlled walking, powerfully regulate the excitability of the somatic afferent paths from forelimb mechanoreceptors (including low threshold cutaneous receptors) to motor cortex. Retention (or enhancement) of responsiveness often occurred (especially for ulnar nerve) around footfall, perhaps reflecting a behavioural requirement for sensory input signalling the quality of the contact established with the restricted surface available for support. (+info)
Postnatal limb bone growth in relation to live weight in pigs from birth to 84 days of age.
(23/1162)
Long limb bones were dissected from 30 Camborough x Canabrid pigs serially slaughtered at birth, 14, 28, 56, and 84 d of age to determine the growth patterns of long limb bones (humerus and radius for the forelimb and femur and tibia for the hindlimb) in relation to live weight from birth to 84 d of age, weighing up to approximately 31 kg. Relationships between individual long limb bone measurements (Y) and live weight or transformations of live weight (X) were evaluated using allometric analyses. Hindlimb bones tended to have higher growth coefficients than forelimb bones (P < .05), suggesting an anterior-posterior gradient of long limb bone growth. However, centripetal gradients of growth for long limb bones were not evidential (P > .05). Greater diameter growth compared with length growth of the long limb bones indicated that the differentiation in growth for diameter and length of the long limb bones was an effective response to the functional requirements of standing, walking, and running following birth. (+info)
Differential patterns of spinal sympathetic outflow involving a 10-Hz rhythm.
(24/1162)
Time and frequency domain analyses were used to examine the changes in the relationships between the discharges of the inferior cardiac (CN) and vertebral (VN) postganglionic sympathetic nerves produced by electrical activation of the midbrain periaqueductal gray (PAG) in urethan-anesthetized, baroreceptor-denervated cats. CN-VN coherence and phase angle in the 10-Hz band served as measures of the coupling of the central oscillators controlling these nerves. The 10-Hz rhythm in CN and VN discharges was entrained 1:1 to electrical stimuli applied to the PAG at frequencies between 7 and 12 Hz. CN 10-Hz discharges were increased, and VN 10-Hz discharges were decreased when the frequency of PAG stimulation was equal to or above that of the free-running rhythm. In contrast, stimulation of the same PAG sites at lower frequencies increased, albeit disproportionately, the 10-Hz discharges of both nerves. In either case, PAG stimulation significantly increased the phase angle between the two signals (VN 10-Hz activity lagged CN activity); coherence values relating their discharges were little affected. However, the increase in phase angle was significantly more pronounced when the 10-Hz discharges of the two nerves were reciprocally affected. Importantly, partialization of the phase spectrum using the PAG stimuli did not reverse the change in CN-VN phase angle. This observation suggests that the increase in the CN-VN phase angle reflected changes in the phase relations between coupled oscillators in the brain stem rather than the difference in conduction times to the two nerves from the site of PAG stimulation. In contrast to the effects elicited by PAG stimulation, stimulation of the medullary lateral tegmental field induced uniform increases in the 10-Hz discharges of the two nerves and no change in the CN-VN phase angle. Our results demonstrate that changes in the phase relations among coupled brain stem 10-Hz oscillators are accompanied by differential patterns of spinal sympathetic outflow. The reciprocal changes in CN and VN discharges produced by PAG stimulation are consistent with the pattern of spinal sympathetic outflow expected during the defense reaction. (+info)