Blastemal kinetics and pattern formation during amphibian limb regeneration.
(9/1162)
To investigate whether the uniqueness of proximal and distal limb regenerates could be attributed simply to differing blastemal growth characteristics, their increase in volume, cell number and cell-cycle times were determined. With respect to these parameters proximal and distal blastemas were identical and, furthermore, no evidence could be found for the existance of separate growth zones such as an apical proliferation centre or a progress zone within the blastema. It was therefore concluded that level-specific properties of the blastemal cells play the major role in determining the structure of the regenerate, not their growth characteristics. The only discernible difference was in the cell number within the two types of blastema at the onset of cartilage redifferentiation - proximal regenerates had 60% more cells. Thus it seems that the larger the pattern to be regenerated (the more proximal the amputation plane), the larger the primordium within which that pattern first appears. These two conclusions are discussed in relation to current theories of pattern formation during limb regeneration and development and a few way of envisaging the regeneration of pattern is described. (+info)
Direct exposure of mouse embryonic limb-buds to 5-bromodeoxyuridine in vitro and its effect on chondrogenesis: increasing resistance to the analog at successive stages of development.
(10/1162)
The inhibitory effect of 5-bromodeoxyuridine (BudR) - an analog of thymidine - on embryonic mouse limb-buds was studied in vitro employing an organ-culture system. The effect was found to be dose-related and also depended on the developmental stage of the donor embryos. Limbs at an early stage development (early 11 th-day embryos, somite stage 26-29) were extremely sensitive to the analog. Treatment with low levels (2-4 mug/ml) and for a relatively short period of time in cluture (2-3 days) completely and irreversibly suppressed chondrogenesis in these explants. Limbs from older embryos (somite stage 40 and up) were found to be much less sensitive to the inhibitory effect of the drug; a prolonged exposure to a much higher dose (100-150 mug/ml) resulted in an incomplete suppression of chondrogesesis. Only a 20% inhibition was observed in the cultures of limbs from mid-13th-day mouse embryos. After continuous growth in vitro, the limbs became progressively resistent to the analog and towards the end of the culture period had become refractory to the drug. The time of complete insensitivity appeared earlier in the cures of the limbs taken from older embryos than in the explants of youngerlimbs. These studies show that as limbs continue to differentiate in vivo or in vitro, they become increasingly resistent to the inhibitory effect of BudR in at least as far as the effect on the process of chondrogenesis is concerned. It is suggested that the relative sensitivity or insensitivity to the inhibitory effect of BudR may prove to be a useful parameter in evaluating the developmental stage of an organ. (+info)
Retinoid signaling is required for chondrocyte maturation and endochondral bone formation during limb skeletogenesis.
(11/1162)
Retinoids have long been known to influence skeletogenesis but the specific roles played by these effectors and their nuclear receptors remain unclear. Thus, it is not known whether endogenous retinoids are present in developing skeletal elements, whether expression of the retinoic acid receptor (RAR) genes alpha, beta, and gamma changes during chondrocyte maturation, or how interference with retinoid signaling affects skeletogenesis. We found that immature chondrocytes present in stage 27 (Day 5.5) chick embryo humerus exhibited low and diffuse expression of RARalpha and gamma, while RARbeta expression was strong in perichondrium. Emergence of hypertrophic chondrocytes in Day 8-10 embryo limbs was accompanied by a marked and selective up-regulation of RARgamma gene expression. The RARgamma-rich type X collagen-expressing hypertrophic chondrocytes lay below metaphyseal prehypertrophic chondrocytes expressing Indian hedgehog (Ihh) and were followed by mineralizing chondrocytes undergoing endochondral ossification. Bioassays revealed that cartilaginous elements in Day 5.5, 8.5, and 10 chick embryo limbs all contained endogenous retinoids; strikingly, the perichondrial tissues surrounding the cartilages contained very large amounts of retinoids. Implantation of beads filled with retinoid antagonist Ro 41-5253 or AGN 193109 near the humeral anlagens in stage 21 (Day 3.5) or stage 27 chick embryos severely affected humerus development. In comparison to their normal counterparts, antagonist-treated humeri in Day 8.5-10 chick embryos were significantly shorter and abnormally bent; their diaphyseal chondrocytes had remained prehypertrophic Ihh-expressing cells, did not express RARgamma, and were not undergoing endochondral ossification. Interestingly, formation of an intramembranous bony collar around the diaphysis was not affected by antagonist treatment. Using chondrocyte cultures, we found that the antagonists effectively interfered with the ability of all-trans-retinoic acid to induce terminal cell maturation. The results provide clear evidence that retinoid-dependent and RAR-mediated mechanisms are required for completion of the chondrocyte maturation process and endochondral ossification in the developing limb. These mechanisms may be positively influenced by cooperative interactions between the chondrocytes and their retinoid-rich perichondrial tissues. (+info)
Morphological clues from multilegged frogs: are retinoids to blame?
(12/1162)
Morphological analysis was performed on multilegged deformed frogs representing five species from 12 different localities in California, Oregon, Arizona, and New York. The pattern of duplicated limbs was consistent with mechanical perturbation by trematode infestation but not with the effects of retinoids. (+info)
Organization of somatosensory areas I and II in marsupial cerebral cortex: parallel processing in the possum sensory cortex.
(13/1162)
Organization of somatosensory areas I and II in marsupial cerebral cortex: parallel processing in the possum sensory cortex. Controversy exists over the organization of mammalian thalamocortical somatosensory networks. An issue of particular contention is whether the primary and secondary somatosensory areas of cortex (SI and SII) are organized in a parallel or serial scheme for processing tactile information. The current experiments were conducted in the anesthetized brush-tail possum (Trichosurus vulpecula) to determine which organizational scheme operates in marsupials, which have taken a quite different evolutionary path from the placental species studied in this respect. The effect of rapid reversible inactivation of SI, achieved by localized cortical cooling, was examined on both evoked potential and single neuron responses in SII. SI inactivation was without effect on the amplitude, latency, and time course of SII-evoked potentials, indicating that the transient inputs responsible for the SII-evoked potential reach SII directly from the thalamus rather than traversing an indirect serial route via SI. Tactile responsiveness was examined quantitatively before, during, and after SI inactivation in 16 SII neurons. Fourteen were unchanged in their responsiveness, and two showed some reduction, an effect probably attributable to the loss of a facilitatory influence exerted by SI on a small proportion of SII neurons. The temporal precision and pattern of SII responses to dynamic forms of mechanical stimuli were unaffected, and temporal dispersion in the SII response bursts was unchanged in association with SI inactivation. In conclusion, the results establish that, within this marsupial species, tactile inputs can reach SII directly from the thalamus and are not dependent on a serially organized path through SI. A predominantly parallel organizational scheme for SI and SII operates in this representative of the marsupial order, as it does in a range of placental mammals including the cat and rabbit, the tree shrew and prosimian galago, and at least one primate representative, the marmoset monkey. (+info)
Pharmacokinetic advantage of intra-arterial cyclosporin A delivery to vascularly isolated rabbit forelimb. I. Model development.
(14/1162)
Effective antirejection therapy with minimal systemic morbidity is required if limb transplantation is to become a clinical reality. We investigated whether i.a. infusion of cyclosporin A (CSA) into the vascularly isolated rabbit forelimb will distribute drug homogeneously to the tissues and produce higher local drug levels than same-dose i.v. treatment, thereby improving the therapeutic index. CSA 4.0 mg/kg/day was infused continuously via osmotic minipump into either the right brachial artery (i.a. group) or jugular vein (i.v. group) of New Zealand rabbits. Ligation of all muscles at the right mid-arm level was performed in the i.a. group to eliminate collateral circulation and simulate allografting, while leaving bone and neurovasculature intact. On day 6, CSA concentrations were measured in skin, muscle, bone, and bone marrow samples taken from different compartments of the right and left forearms in the i.a. group and right forearm only in the i.v. group. There were no significant differences between compartmental CSA levels in all tissues examined on the locally treated, right side during i.a. infusion, indicating that drug streaming from the catheter tip is not occurring in our model. During i.a. infusion, mean CSA concentrations were 4- to 7-fold higher in the right limb than in the left limb in all four tissues examined. Tissue CSA levels in the left limb were equivalent to those achieved during i.v. infusion, but CSA concentrations in blood, kidney, and liver were higher during i.a. infusion. These favorable, preliminary, single-dose pharmacokinetic results warrant further investigation in our novel rabbit model. (+info)
Pharmacokinetic advantage of intra-arterial cyclosporin A delivery to vascularly isolated rabbit forelimb. II. Dose dependence.
(15/1162)
A vascularly isolated rabbit forelimb model simulating conditions of composite tissue allografting was used to determine the regional pharmacokinetic advantage achievable in extremity tissue components during i.a. cyclosporin A (CSA) administration. CSA was infused continuously via osmotic minipump into the right brachial artery of New Zealand rabbits at multiple doses ranging from 1.0 to 8.0 mg/kg/day. On day 6, CSA concentrations were measured in aortic whole blood, as well as in skin, muscle, bone, and bone marrow samples from both right and left forelimbs. The variation of right-sided mean CSA concentrations with dose was tissue dependent and saturable in the case of skin and bone, whereas left-sided tissue concentrations correlated significantly with systemic blood levels. At 1.0 mg/kg/day, there were no significant differences between right and left mean CSA concentrations for all four tissues examined. However, with a doubling of the i.a. dose, huge increases in local tissue CSA concentrations were produced with only very modest increases in systemic whole-blood and tissue drug levels, resulting in a 4-fold regional advantage (right/left ratio of CSA concentrations) in bone and bone marrow, 7-fold in muscle, and 14-fold in skin. With further dose increases to 8.0 mg/kg/day, the regional advantage decreased to 4-fold in skin, increased to 9-fold in bone marrow, remained relatively constant in bone, and initially decreased and then increased to 9-fold in muscle. These favorable pharmacokinetic results suggest that reduced, local doses of CSA might be useful in preventing extremity composite tissue allograft rejection with decreased systemic drug exposure. (+info)
Pharmacokinetics of intra-arterial delivery of tacrolimus to vascularly isolated rabbit forelimb.
(16/1162)
A vascularly isolated rabbit forelimb model simulating conditions of composite tissue allografting was used to determine the regional pharmacokinetic advantage achievable in extremity tissue components during i.a. tacrolimus (FK506) administration. FK506 was infused continuously via osmotic minipump into the right brachial artery of New Zealand rabbits at 0.05, 0.1, and 0.2 mg/kg/day. On day 6, FK506 concentrations were measured in aortic whole blood, heart, lung, liver, kidney, spleen, and fat, as well as in skin, muscle, bone, and bone marrow samples from both right and left forelimbs. The relative tissue concentrations of FK506 in descending order were [spleen approximately lung approximately kidney] > [heart approximately skin approximately muscle] > [fat approximately bone marrow] > [liver approximately bone approximately blood]. In marked contrast to previous results with i.a. cyclosporin A infusion, only a minimal regional advantage of local FK506 delivery (mean right/left concentration ratios 1.0-1.4) was obtained in all forearm tissues over the dose range studied. For each limb tissue, left-sided FK506 concentrations significantly correlated with systemic blood levels, and the left-sided tissue-to-whole-blood concentration ratio did not vary significantly with dose. We conclude that FK506 is pharmacokinetically inferior to cyclosporin A for continuous i.a. administration to the vascularly isolated rabbit forelimb, and hypothesize that this difference is the result of differences in the distribution of each drug within whole blood. Our findings suggest that, despite its demonstrated efficacy in experimental and clinical transplantation, FK506 would not be an appropriate immunosuppressant to deliver via the i.a. route for prevention of limb allograft rejection. (+info)