Retinoids as endogenous components of the regenerating limb and tail

The role of retinoids (vitamin A derivatives) in limb and tail regeneration is the focus of this review. The dramatic effects of excess retinoids on regeneration are considered, which consist of the respecification of each of the cardinal axes of the limb and the transformation of tail regenerates into limbs. The evidence that retinoids are endogenous components of the regeneration blastema and are required for normal regeneration is then addressed. This evidence consists of the fact that various techniques can detect endogenous retinoic acid in the blastema and that retinoic acid receptors are present, and an individual function in regeneration has been established for each of three receptors. Finally, experiments are described in which retinoic synthesis has been inhibited, and this results in the inhibition of both limb development and limb and tail regeneration.     

The cortical somatotopic map and phantom phenomena in subjects with congenital limb atrophy and traumatic amputees with phantom limb pain

The extent of the cortical somatotopic map and its relationship to phantom phenomena was tested in five subjects with congenital absence of an upper limb, four traumatic amputees with phantom limb pain and five healthy controls. Cortical maps of the first and fifth digit of the intact hand, the lower lip and the first toe (bilaterally) were obtained using neuroelectric source imaging. The subjects with congenital upper limb atrophy showed symmetric positions of the left and right side of the lower lip and the first toe, whereas the traumatic amputees with pain showed a significant shift (about 2.4 cm) of the cortical representation of the lower lip towards the hand region contralateral to the amputation side but no shift for the toe representation. In healthy controls, no significant hemispheric differences between the cortical representation of the digits, lower lip or first toe were found. Phantom phenomena were absent in the congenital but extensive in the traumatic amputees. These data confirm the assumption that congenital absence of a limb does not lead to cortical reorganization or phantom limbs whereas traumatic amputations that are accompanied by phantom limb pain show shifts of the cortical areas adjacent to the amputation zone towards the representation of the deafferented body part.     

Critical limb ischemia

Chronic critical limb ischemia is defined as ischemia which generally endangers the distal part of a limb. If the ischemia is unrelenting, there is a high risk that amputation will be required. These critical limbs were amputated as an initial treatment long ago. Now it becomes clear that disabling patients have very poor quality of life after major amputation, and ischemic leg produces noxious substances which circulates whole body. So that primary amputation should not be a first choice for the most of the cases. And excellent techniques or new surgical materials bring successful vascular reconstruction to critical limb ischemia. Since we have not a clear definition, European and US criterion seems to be useful for a while. Macrocirculation is represented by ankle systolic pressure and microcirculation by transcutaneous oxygen pressure as well. Its value of les than 10 mmHg means urgent critical condition. Main surgical repairs include below knee bypass using autovein or fine profundaplasty with or without PTA. Usually patients should receive general cares because of high risks.     

Is it possible to predict limb viability in complex Gustilo IIIB and IIIC tibial fractures? A comparison of two predictive indices

The patient with severe lower limb trauma presents a management dilemma; whether to amputate primarily or to attempt limb salvage. In recent years, many predictive indices have been published which purport to identify limbs which are non-viable. We retrospectively applied two recently described indices, the Mangled Extremity Severity Score (MESS) and the Limb Salvage Index (LSI), to 54 limbs in 50 patients with either Gustilo IIIB or IIIC complex tibial fractures. There were 22 amputations (40.7 per cent) in the series. The mean MESS score in the limb salvage group was 3.8 (range 2-10), and the mean MESS score in the amputation group was 7.7 (range 4-13) (P < 0.0001). The mean LSI score in the limb salvage group was 3.6 (range 3-8), and the mean LSI score in the amputation group was 6.9 (P < 0.01). However, in the group with MESS scores > 7 (which recommends amputation), there were three limbs which were salvaged with acceptable functional outcome. Similarly, in those with LSI scores > 6 (which recommends amputation), there were seven limbs successfully salvaged. A MESS > 7 offered a greater relative risk of amputation (9.2) than a LSI score > 6 (5.3). We found both indices of use in predicting limb salvage and functional outcome. However, neither is sufficiently accurate to be considered absolutely reliable in clinical practice.     

Effects of delta9-tetrahydrocannabinol on neurotransmitter accumulation and release mechanisms in rat forebrain synaptosomes

Previous studies involving the end-to-end fusion of the forelimbs of the adult newt have demonstrated that new limbs can regenerate from the transected ends of proximo-distally reversed limb segments. The limb regeneration could only have been initiated by nerve fibers of contralateral origin. The purpose of the present study is to describe histologically the manner in which nerve fibers of contralateral origin regenerate through the junction of fused limbs into the opposite limb. The first sign of nerve regeneration into the opposite limb was observed at eight days post fusion. The nerves crossed over into the opposite, originally denervated limb in a highly dispersed manner. These nerve fibers eventually aggregated, however, either under the skin or within persisting nerve trunks. By 19 days post fusion the nerve fibers had reached the elbow region of the originally denervated limb and by 25 days they were seen at the most proximal extent of the limb. The diameters of the axons seemed smaller than the diameters of regenerating axons observed in non-fused newt forelimbs.     

The prevalence of phantom limb experience in amputees.

Reviewed the records of 716 amputees who utilized Canadian prosthetic facilities to determine the prevalence of phantom limb pain or sensation and the possible influence of demographic variables on their presence. The prevalence of phantom pain (62.4%) and phantom sensation (84.1%) tended to confirm recent surveys in other nationalities. Years since amputation was the only variable found to influence the report of either pain or sensation. Ss who lost their limbs prior to 1960 reported significantly more pain (73.3%) than Ss who lost their limbs after 1976 (51.1%). There was also a slight tendency for older Ss to report phantom limb pain more often than younger Ss. Findings suggest that the incidence of phantom limb pain is declining. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Lower limb amputation. 3: The role of the nurse

This article, the third in a series on lower limb amputation, examines the role of the nurse in the pre- and postoperative care of patients undergoing amputation. The nurse has an integral role not only in providing care but also in liaising with other members of the multidisciplinary team in order to ensure that the person undergoing the amputation feels prepared for both the operation and discharge home. Amputation may greatly distort an individual's vision of him/herself as a person, a partner and a parent; ways in which this distortion can be minimized are explored.     

Early chick limb cartilaginous elements possess polarizing activity and express hedgehog-related morphogenetic factors

Skeletal patterning and morphogenesis in the developing limb are thought to be regulated by instructive factors and cues from the zone of polarizing activity (ZPA), the apical ectodermal ridge (AER), and the dorsal ectoderm. However, the activities of the ZPA and AER dwindle early in embryogenesis and soon after ceases, when in fact the proximal skeletal elements are still rudimentary in structure and the more distal ones are yet to become recognizable. Thus, we asked whether the chondrocytes emerging within each mesenchymal condensation may themselves start expressing properties similar to those of ZPA and/or AER and, in so doing, may bring skeletal development to completion. Indeed, we found that the cartilaginous, but not precartilaginous, tissues in early chick limbs possess ZPA-like properties. They expressed an endogenous factor related to Sonic hedgehog (Shh), most likely Indian hedgehog (Ihh), and when fragments were grafted to the anterior margin of host stage 16-20 chick wing buds, they induced supernumerary skeletal elements (polarizing activity). The acquisition of polarizing activity by the cartilaginous structures followed clear proximo-to-distal and posterior-to-anterior routes. Thus, (1) stage 25 cartilaginous humerus had polarizing activity while stage 25 prospective radius did not, (2) posteriorly-located stage 29 ulna had stronger activity than anteriorly-located stage 29 radius, and (3) ulna's diaphysis had stronger activity at stage 29 than 31 while radius's diaphysis was stronger at stage 31 than 29. Prior to inducing extra digit formation, the cartilaginous grafts induced Hoxd-12 and Hoxd-13 gene expression in adjacent competent mesenchymal tissue. Strikingly, the cartilaginous grafts activity also expression of Shh and polarizing activity in adjacent mesenchyme, which ZPA grafts cannot do; thus, the cartilaginous structures displayed activities "upstream" of those of the ZPA. The results support our hypothesis that chondrocytes may themselves direct skeletal morphogenesis. In so doing and as a result of their inductive activities, the cells may also have an important role in the completion of limb patterning and morphogenesis.     

Early branching eukaryotes?

The effect of hyperbaric oxygen (HBO) on ischemic muscle tissue pH was evaluated continuously. The hind limbs of male Sprague-Dawley rats (N=11, both groups) were amputated and stored in room air (20.1% oxygen [O2], 1.0 ATM, 24 degrees C) or in HBO (100% O2, 2.9 ATM, 24 degrees C) for 240 minutes. Rat muscle tissue pH was continuously monitored with a micro-pH electrode following amputation. There was no significant difference between the average starting tissue pH of control and treated limbs (p=0.45). At 240 minutes of ischemia the control group tissue pH decreased 0.80 pH units whereas the treatment group decreased 0.68 pH units (p < 0.05). The tissue pH of control limbs declined 30.7 times faster than treated limbs during the first 36 minutes of ischemia (p < 0.05). From 36 to 240 minutes the rates of acidosis were similar and did not differ significantly (p=0.46). In a separate study, male Sprague-Dawley rats were anesthetized with pentobarbital and ketamine. Aortic arterial blood gases were obtained at 5 minutes (N=8) and 15 minutes (N=8) postanesthesia. The average serum pH, carbon dioxide, oxygen, and bicarbonate levels remained within normal limits in both groups and did not differ significantly (p > 0.05 for all parameters). Anesthesia produced no serum respiratory or metabolic acidosis and did not contribute to the initial ischemic tissue pH. These results suggest that HBO delays the progression of metabolic acidosis in this amputated limb model. This is further supporting evidence for the tissue-preserving effect of oxygen when delivered in hyperbaric conditions. However, the clinical application of this technique may be limited due to the difference in the volume of tissue presented for major limb replantation and the short window of beneficial effects.     

Reduction of ischemic reperfusion edema with corticotropin-releasing factor (CRF) in rat hind limb replantation

Corticotropin-releasing factor (CRF), a peptide neurotransmitter, is suggested as a novel agent to reduce reperfusion edema following ischemia. In a rat hind limb replantation model, animals underwent amputation followed by 2.5 hours of ischemia with replantation and 2 hours of reperfusion. Animals were randomized to seven groups: a nonischemic control group and six experimental groups receiving treatment prior to reperfusion with (1) saline control, (2) alpha 9,41-CRF-a CRF receptor blocking agent (98 micrograms/per kilogram), (3) subcutaneous CRF (320 micrograms/per kilogram), (4) intravenous CRF (80 micrograms/per kilogram), (5) alpha-CRF and subcutaneous CRF, and (6) alpha-CRF and intravenous CRF. Comparison of preischemic amputated limb weight with weight after ischemia and reperfusion showed a reproducible and significant gain in limb weight after 2 hours (p = 0.004). A significant reduction in limb weight gain (49%) was achieved with both subcutaneous (p < 0.04) and intravenous CRF (p < 0.036). With the dose used in this model, alpha 9,41 CRF attenuated but did not completely block the effects of intravenous or subcutaneous CRF.     

Sympathetic blockade of isolated rat hindlimbs by intra-arterial guanethidine: the effect on blood flow and arterial-venous shunting

In order to improve the understanding of the role of sympathetic nerve degeneration in reimplantation failure, the hindlimbs of eight rats (Group I) underwent near-complete amputation. The soft tissues of the hindlimb were transected at the proximal thigh with the femoral artery, vein and femur left intact. The femoral vessels were clamped and guanethidine was infused into a branch of the femoral artery of the right leg of each animal, while saline was injected into the left leg. The clamps were removed after 15 minutes. A baseline preoperative injection of radiolabeled microspheres was made, and subsequent injections at 6, 12, 18, and 24 hours postoperation. Twelve rats (Group II) were then used to assess the amount of arterial-venous shunting preoperatively (n = 6) and at 18 hours postoperation (n = 6), by venous sampling. Blood flow to both limbs increased postoperation, but there was significantly more flow in the guanethidine treated limb at 18 and 24 hours postoperation. The amount of shunting was approximately 50% in both limbs at 18 hours, as compared to 10% preoperation. These results highlight the potential benefit of guanethidine and other sympathetic blocking agents in reimplantation to increase blood flow, decrease tissue ischemia and increase anastomotic patency rates. They also suggest that sympathetic nerve degeneration did not affect the volume of arterial-venous shunting in this model, but the difference in blood flow was likely due to arteriolar vasospasm. Further study is needed to elucidate the clinical significance of sympathetic nerve degeneration in reimplantation failure.     

Expression of regeneration-associated cytoskeletal proteins reveals differences and similarities between regenerating organs

The unique events which allow regeneration of an entire organ to occur are formation of a specialized wound epidermis and accumulation of progenitor cells (blastemal cells) at the amputated surface to form a blastema. In order to identify some of the molecular events underlying the early stages of the regenerative process which are either common to different systems or specific to one of them, we have investigated whether molecules which are induced in limb blastemas are also expressed in skin repair and during regeneration of other complex body structures (lower jaws, upper jaws, and tails). In addition, we have addressed the issue of the identity of progenitor cells during jaw development and regeneration by analyzing the expression of limb blastemal markers in the developing head and face. We have focused on cytoskeletal components, and particularly on the epidermal keratin NvKII, the simple epithelial keratins 8 and 18 and 22/18, because they are among the few molecules which have been shown to be associated with regeneration in the limb and may play significant roles in various developmental processes. Some important findings emerge from this study: 1) Expression of the epidermal keratin NvKII, unlike that of its mammalian homologue K6, is not simply induced in response to wounding, but is associated with regeneration of specific organs. In fact, NvKII is expressed in regenerating limbs and tails, but not in upper or in lower jaw regenerates, demonstrating the existence of molecular differences in the composition of the wound epidermis in these systems. This, together with the fact that NvKII mRNA is regulated by retinoic acid, which differentially affects patterning of limbs and jaws, argues for distinct inductive abilities of the wound epidermis in different organs. 2) In contrast to the differential expression of the epidermal keratin NvKII, the regeneration-associated cytoskeletal molecules identified in limb blastemal cells are expressed in a similar fashion in jaw and tail blastemas. Therefore, it appears that similar cellular events lead to the establishment of an actively proliferating population of progenitor cells from the stump of different organs. Finally, the mesenchyme of the facial rudiments, unlike that of developing limb buds, expresses simple epithelial keratins. Thus, it appears that mesenchymal progenitor cells of developing and regenerating jaws are alike in regard to their intermediate filament content, and this may be related to nerve-dependent growth control of progenitor cells in different developing and regenerating systems.     

Relationship between dose, distance and time in Sonic Hedgehog-mediated regulation of anteroposterior polarity in the chick limb

Anteroposterior polarity in the vertebrate limb is thought to be regulated in response to signals derived from a specialized region of distal posterior mesenchyme, the zone of polarizing activity. Sonic Hedgehog (Shh) is expressed in the zone of polarizing activity and appears to mediate the action of the zone of polarizing activity. Here we have manipulated Shh signal in the limb to assess whether it acts as a long-range signal to directly pattern all the digits. Firstly, we demonstrate that alterations in digit development are dependent upon the dose of Shh applied. DiI-labeling experiments indicate that cells giving rise to the extra digits lie within a 300 microm radius of a Shh bead and that the most posterior digits come from cells that lie very close to the bead. A response to Shh involves a 12-16 hour period in which no irreversible changes in digit pattern occur. Increasing the time of exposure to Shh leads to specification of additional digits, firstly digit 2, then 3, then 4. Cell marking experiments demonstrate that cells giving rise to posterior digits are first specified as anterior digits and later adopt a more posterior character. To monitor the direct range of Shh signalling, we developed sensitive assays for localizing Shh by attaching alkaline phosphatase to Shh and introducing cells expressing these forms into the limb bud. These experiments demonstrate that long-range diffusion across the anteroposterior axis of the limb is possible. However, despite a dramatic difference in their diffusibility in the limb mesenchyme, the two forms of alkaline phosphatase-tagged Shh proteins share similar polarizing activity. Moreover, Shh-N (aminoterminal peptide of Shh)-coated beads and Shh-expressing cells also exhibit similar patterning activity despite a significant difference in the diffusibility of Shh from these two sources. Finally, we demonstrate that when Shh-N is attached to an integral membrane protein, cells transfected with this anchored signal also induce mirror-image pattern duplications in a dose-dependent fashion similar to the zone of polarizing activity itself. These data suggest that it is unlikely that Shh itself signals digit formation at a distance. Beads soaked in Shh-N do not induce Shh in anterior limb mesenchyme ruling out direct propagation of a Shh signal. However, Shh induces dose-dependent expression of Bmp genes in anterior mesenchyme at the start of the promotion phase. Taken together, these results argue that the dose-dependent effects of Shh in the regulation of anteroposterior pattern in the limb may be mediated by some other signal(s). BMPs are plausible candidates.     

Re-examining jaw regeneration in urodeles: what have we learnt?

Urodele amphibians can regenerate not only their limbs and tails, but also their upper and lower jaws rather faithfully. However, relatively few studies of jaw regeneration in amphibians have been carried out, especially in recent years. It is therefore important to reexamine thoroughly this regenerating system, since the advent of sophisticated morphological techniques and the development of molecular approaches offer the promise of renewed and rapid progress in our understanding of complex developmental problems such as this. This paper briefly reviews some of the early research on jaw regeneration, some of the fundamental questions which have been asked and have yet to be answered, and the work we have carried out in order to understand the molecular mechanisms underlying jaw regeneration in the newt, Notophthalmus viridescens. In addition, some aspects of jaw regeneration will be discussed in relation to regeneration of the adult limb.     

Heterogeneity among muscle precursor cells in adult skeletal muscles with differing regenerative capacities

Skeletal muscle has a remarkable capacity to regenerate after injury, although studies of muscle regeneration have heretofore been limited almost exclusively to limb musculature. Muscle precursor cells in skeletal muscle are responsible for the repair of damaged muscle. Heterogeneity exists in the growth and differentiation properties of muscle precursor cell (myoblast) populations throughout limb development but whether the muscle precursor cells differ among adult skeletal muscles is unknown. Such heterogeneity among myoblasts in the adult may give rise to skeletal muscles with different regenerative capacities. Here we compare the regenerative response of a masticatory muscle, the masseter, to that of limb muscles. After exogenous trauma (freeze or crush injuries), masseter muscle regenerated much less effectively than limb muscle. In limb muscle, normal architecture was restored 12 days after injury, whereas in masseter muscle, minimal regeneration occurred during the same time period. Indeed, at late time points, masseter muscles exhibited increased fibrous connective tissue in the region of damage, evidence of ineffective muscle regeneration. Similarly, in response to endogenous muscle injury due to a muscular dystrophy, widespread evidence of impaired regeneration was present in masseter muscle but not in limb muscle. To explore the cellular basis of these different regenerative capacities, we analyzed the myoblast populations of limb and masseter muscles both in vivo and in vitro. From in vivo analyses, the number of myoblasts in regenerating muscle was less in masseter compared with limb muscle. Assessment of population growth in vitro indicated that masseter myoblasts grow more slowly than limb myoblasts under identical conditions. We conclude that the impaired regeneration in masseter muscles is due to differences in the intrinsic myoblast populations compared to limb muscles.     

Incorporation of bromodeoxyuridine in regenerating fin tissue of the goldfish Carassius auratus

We have investigated the pattern of incorporation of 5-bromo-2'-deoxyuridine-5'-monophosphate (BrdU) by proliferating cells during regeneration of the tail fin of Carassius auratus. Fifteen days after amputation, intraperitoneal injection of a single dose of 0.25 mg/g wet weight of BrdU and subsequent immunocytochemical detection on sections revealed groups of replicating cells in the blastema and epidermis at different proximodistal levels. Proliferating blastemal cells were confined to a crowded, compact distal area that lost its replicative capacity laterally, causing the differentiation of scleroblasts, which synthesize the lepidotrichia hemisegments. Proximally, but centrally located, the blastemal cells did not incorporate BrdU and they differentiated giving rise to the mature intraray connective tissue. An independent cell-proliferation process was noted in the epidermis. The distal cap did not proliferate; the lateral faces of the epidermis showed high rates of cell replication in the central layer at every level of the regenerate rays; quiescent cells remained in the superficial layers. The basal epidermal cells did not incorporate BrdU when actinotrichia were present. The possible role of basal epidermal cells in the synthesis of actinotrichia, the contribution of these collagen macrofibrils to the morphogenetic process, and the different pathways of cell differentiation during fin regeneration are discussed.     

Lower limb amputation. 1: indications and treatment

Lower limb amputation is performed predominantly to alleviate acute and chronic limb ischaemia caused by vascular disease, poorly controlled diabetes or, occasionally, infection. Atherosclerosis is the primary cause of chronic arterial ischaemia and the most common reason for amputation. The vascular nurse has an important role in reducing the need for amputation, by providing information on health promotion and illness prevention to patients with vascular insufficiency to halt progression to amputation. This is the first of four articles focusing on lower limb amputation. It examines the indications for lower limb amputation in detail, and briefly outlines other treatment options including revascularization techniques.     

Peripheral arterial insufficiency. Experiences from 229 operated limbs

The results of arterial reconstructions in 229 limbs in 177 patients were studied. Intermittent claudication resistant to physical training was indication for surgery in 82 cases and threatening gangrene in 147 cases. In the claudication group the long-term result was good in 77%, the reocclusion frequency was 21%, and the amputation rate was 2%. No operative mortality was encountered. In the gangrene group the long-term result was good in 40%, the reocclusion freuqency was 25%, the amputation rate was 24%, and theoperative mortality was 11%. Limb salvage rate was 65%. The results indicated that preoperative physical training did not jeopardice the operative results, but in most cases made surgical treatment unnecessary. In cases with threatening or beginning gangrene an aggressive attitude towards vascular reconstruction resulted in a substantial limb salvage.     

The development and enhancement of the collateral circulation in an animal model of lower limb ischaemia

In an animal model of hind limb ischemia we documented the levels of endogenous basic fibroblast growth factor (bFGF) in control and ischaemic hind limbs, and evaluated the response to the administration of exogenous recombinant bFGF and heparin. Variations in this model were tested for their ability to alter the development of the collateral circulation. Recovery after acute arterial occlusion was significantly delayed by immediate bilateral mirror-image arterial ligations, when compared with either unilateral arterial ligation or delayed contralateral ligations performed after 2 months. If the major veins were also occluded all limbs developed gangrene, tissue loss and a marked delay in the recovery of blood flow, while none of the animals with unilateral arterial ligations developed gangrene. This indicates that the recovery in blood flow during the acute phase in this model is dependent on collateral vessels from the contralateral iliac artery and that major venous occlusion impedes the development of collateral vessels. Lumbar sympathectomy did not alter the recovery of blood flow after arterial occlusion, suggesting that collateral blood flow is not significantly influenced by autonomic neural supply. Following arterial occlusion there was a ten-fold increase in the levels of endogenous bFGF in all ischaemic muscle groups. Intramuscular implantation of bFGF in heparin-sepharose pellets at the time of arterial ligation markedly enhanced the blood flow for 3 weeks compared with untreated ischaemic limbs. A further increment in blood flow occurred if an additional dose of bFGF was administered 4 weeks after ligation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tissue blood flow distribution and the effect of chronic vascular catheterization in the hind limb of the fetal lamb

In six chronically instrumented fetal lambs, hind-limb skin, bone, and muscle comprised 22.5 +/- 1.3, 35.3 +/- 1.6, and 42.3 +/- 1.1% of total limb weight, respectively. As estimated using radionuclide-labeled microspheres, blood flow to these tissues averaged 30.4 +/- 4.9, 30.1 +/- 3.3, and 14.0 +/- 3.1 mL.min-1.100 g-1, respectively, and they received 29.5 +/- 3.3, 45.3 +/- 3.6, and 25.2 +/- 4.5% of total limb blood flow. Thus, muscle has a lower blood flow in relation to its weight in comparison with the other tissues, while bone receives the largest fraction of hind-limb blood flow. The higher perfusion rate to bone may by due to a high rate of hematopoiesis in late gestation, whereas muscle flow may be lower than that reported immediately after birth because of limited limb movement and lack of shivering thermogenesis. There were no significant differences in tissue weights between the limb in which femoral arterial and lateral tarsal venous catheters were implanted (nonstudy limb) and the leg that had smaller diameter catheters placed in the pudendoepigastric artery and vein (study limb). However, nonstudy limb blood flow was 13.4 +/- 1.8% less than in the study limb, although the flow distribution to hind-limb tissues was not different between the two limbs. This suggests that the longer, larger diameter catheters inserted into the nonstudy limb had an adverse effect on hind-limb blood flow but not on overall limb growth or blood flow distribution. More attention should be paid to the effects of chronic fetal vascular catheterization on the tissues or organs normally perfused by the catheterized vessel.