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[Role of the nervous system during the regeneration of an arm in a starfish: Asterina gibbosa Penn. (Echinodermata, Asteriidae)].

In Asterina gibbosa regeneration of a cut-off arm tip cannot occur if the radial nerve has been removed. This regeneration needs the presence of the oral nerve ring connected with the radial nerve intact up to the level of the wound tissues. The oral nerve ring can be considered as a central nervous system. Exposure of the radial nerve to irradiation induces a delay in regeneration. These data suggest there is a stimulation from the oral nerve ring up to the wound tissues. When the tissues involved in regeneration, except for the radial nerve, have been destroyed by irradiation, regeneration is inhibited. Stimulation by a healthy radial nerve is necessary to maintain differentiation and growth of the blasternal cells. The amount of blasternal growth and differentiation depends on the time when the radial nerve ceases to stimulate regeneration. The action of the nervous system is needed throughout the whole course of regeneration.

Animals↗

[Muscle regeneration: the effect of the basal lamina, size of the lesion and inflammatory response in C57BL10/ScSn mice].

INTRODUCTION: The process of muscle regeneration is influenced by many factors. There is little data in the literature regarding the influence of the size of the lesion and inflammatory response on this process. Also, the concept of the integrity of the basal lamina as an essential factor for correct regeneration has recently been questioned. OBJECTIVES: To investigate the participation of these three variables in the process of muscle regeneration and its possible interactions. METHODS: 54 B57BL10/ScSn mice had their gastrocnemius muscle groups injected with glycerol, collagenase glycerol and Chinese ink. The mice were killed at different times following injection. Serial sections 10 mm thick were analyzed using histological and immunohistochemical techniques. RESULTS: By 28 days post injection no difference was observed between mice with intact and altered basement membranes. The lesions with a large number of contiguous necrotic fibres regenerated more slowly. Regeneration was centripetal in these lesions. The inflammatory response depended basically on the type of substance injected. The intensity of the inflammatory response was not positively correlated with increased rate of regeneration. CONCLUSIONS: Alteration or fragmentation of the basal lamina and the size of the lesions seen in this study did not impede complete repair in C57BL10/ScSn mice. Further studies are necessary to establish the basic role played by the inflammatory response in muscular regeneration.

Animals↗

The role of c-fos in cell death and regeneration of retinal ganglion cells.

PURPOSE: To investigate the effect of c-fos on apoptotic cell death and regeneration of damaged retinal ganglion cells (RGCs) in tissue culture of retinal explants. METHODS: Retinas from transgenic mice carrying the exogenous c-fos gene under the control of the interferon (IFN)-alpha/beta inducible Mx-promoter (Mx-c-fos), c-fos-deficient mice, and littermate control mice were dissected and cultured in a three-dimensional collagen gel culture system, followed by an analysis of TdT-dUTP terminal nick-end labeling (TUNEL) staining and measurement of neurites that emerged from explants. RESULTS: Compared with littermate control mice, Mx-c-fos transgenic animals showed a higher ratio of TUNEL positivity in the RGC layer from early in the culture period that correlated with the small number of regenerating neurites. In contrast, the c-fos-null mutated mice showed a still-lower ratio of TUNEL-positive cells. Nevertheless, the number of regenerating neurites was significantly lower in the initial phase, although the drastic increase in density of neurite regeneration was observed in the late period of culture. CONCLUSIONS: These findings suggest that c-fos is involved in both apoptotic cell death and regeneration of damaged RGCs. Elucidation of the precise c-fos-mediated cascade involved in RGC apoptosis and regeneration is significant in realizing neuronal survival and regeneration.

Animals↗

Pre-degenerated peripheral nerve extracts applied to the proximal stump of transected sciatic nerve enhance both regeneration and autotomic behavior in rats.

BACKGROUND: Peripheral nerve regeneration after traumatic injury and standard repair with a nerve autograft is often incomplete and results in neuropathic pain. Peripheral nerve extracts applied to the proximal nerve stump by means of autologous, dead-ended connective tissue chambers (deCTC) are known to accelerate the rate of axon regeneration. This study tested if such extracts would influence autotomy, which is a behavioral measure of neuropathic pain in animal models. MATERIAL/METHODS: The study was performed on Wistar rats. DeCTCs, filled with postmicrosomal fraction obtained from 7-day-predegenerated nerves (7D group) or fibrin (F group), were implanted into transected sciatic nerve. In the control group (C), sciatic nerve was transected and its distal part was removed. The self-mutilation behavior in rats was assessed daily for seven weeks after sciatic nerve transection. The onset as well as intensity of autotomy was measured. The progress of regeneration was assessed under light microscopy and histochemistry. RESULTS: The earliest onset and greatest severity of autotomy were found in the 7D group. Regeneration progress was also highest in this group. In the F group, we found weaker regeneration and less autotomy. The control group showed no regeneration, but more autotomy than the F group. CONCLUSIONS: Intensified autotomy may be the price paid for enhanced peripheral nerve regeneration after application of growth-promoting substances.

Animals↗

[Expression of regulatory homeobox genes during retina regeneration in adult newts].

We studied molecular-genetic mechanisms of retina regeneration in amphibians and, specifically, expression of the homeobox genes Pax6, Prox1, and Six3 in normal development and during retina regeneration in the newt. Based on the structural analysis of genes in closely related amphibian species, primers were constructed that flank certain regions of these genes. PCR fragments of calculated length were obtained. The affiliation of PCR products to the above genes was confirmed by sequencing. A comparative PCR analysis of expression of Pax6, Prox1, and Six3 was carried out in the native and regenerating newt retina, which allowed estimation of the level of expression. cDNA libraries obtained from the native and regenerating retina were used as templates. The libraries were preliminary standardized according to glyceraldehydes-3-phosphate dehydrogenase, an enzyme of general cell metabolism. The genes we studied were expressed in both native and regenerating retina. The level of Pax6 and Prox1 expression increased during regeneration, while that of Six3 decreased. The decrease in the level of Six3 expression could be due to antagonistic interrelations of Prox1 and Six3. The changed level of Prox1 and Six3 expression is a new fact and requires further studies. The interactions between these and other regulatory genes and localization of their expression in the cells of native and regenerating retina will be studied using in situ hybridization and immunohistochemistry.

Age Factors↗

Macrophage-derived factors stimulate optic nerve regeneration.

After optic nerve injury in mature mammals, retinal ganglion cells (RGCs) are normally unable to regenerate their axons and undergo delayed apoptosis. However, if the lens is damaged at the time of nerve injury, many RGCs survive axotomy and regenerate their axons into the distal optic nerve. Lens injury induces macrophage activation, and we show here that factors secreted by macrophages stimulate RGCs to regenerate their axons. When macrophages were activated by intravitreal injections of Zymosan, a yeast cell wall preparation, the number of RGC axons regenerating into the distal optic nerve was even greater than after lens injury. These effects were further enhanced if Zymosan was injected 3 d after nerve crush. In a grafting paradigm, intravitreal Zymosan increased the number of RGCs that regenerated their axons through a 1.5 cm peripheral nerve graft twofold relative to uninjected controls and threefold if injections were delayed 3 d. In cell culture, media conditioned by activated macrophages stimulated adult rat RGCs to regenerate their axons; this effect was potentiated by a low molecular weight factor that is constitutively present in the vitreous humor. After gel-filtration chromatography, macrophage-derived proteins > or =30 kDa were found to be toxic to RGCs, whereas proteins <30 kDa reversed this toxicity and promoted axon regeneration. The protein(s) that stimulated axon growth is distinct from identified polypeptide trophic factors that were tested. Thus, macrophages produce proteins with both positive and negative effects on RGCs, and the effects of macrophages can be optimized by the timing of their activation.

Animals↗

Bone regeneration by recombinant human bone morphogenetic protein-2 around immediate implants: a pilot study in rats.

PURPOSE: Difficulties relating to bone regeneration that complicate immediate implant placement include buccal and/or lingual fenestrations, primary anchorage of the implants, and the need for protection from functional loading during the osseointegration period. The objective of this pilot study was to evaluate bone regeneration by recombinant human bone morphogenetic protein-2 (rhBMP-2) around immediate implants placed in maxillary sockets in rats. MATERIALS AND METHODS: A total of 16 cylindric 0.8 x 1.8-mm commercially pure, solid titanium Implants were placed immediately after gentle extraction of the maxillary first molar teeth of 8 male Wistar rats. The sockets were randomly divided into 3 groups: group 1 (n = 6) received rhBMP-2 with polylactic acid/polyglycolic acid copolymer-coated gelatin sponge carrier; group 2 (n = 5) received only the carrier; and group 3 (n = 5) received no grafting materials following placement The rats were euthanized at 90 days postsurgery for microscopic analysis. RESULTS: In group 1, the implant body remained submerged completely, including the coronal part, which was fully covered by a significant amount (30% of total height) of regenerated cortical bone, even though the implant could easily be pulled out by a tweezer at the time of placement. Close approximation between the implant surface and regenerated bone could also be detected, indicating good bone-to-implant contact. In contrast, only peri-implant bone regeneration occurred in group 2, and an approximate 0.3-mm coronal part of the implant remained exposed. When no grafting materials were used (group 3), almost one third of the total length of the implant was exfoliated out of the socket when no grafting materials were used. DISCUSSION AND CONCLUSIONS: Based on previous study and data from 16 sockets of the present study, it could be concluded that rhBMP-2 facilitated the regeneration of bone around immediate implants. In particular, the bone covering the coronal part could have been regenerated shortly after surgery, which helped to maintain the implant body inside the socket during the integration period in rats.

Animals↗

[Proteomics analysis and biochemical activity detection on facial nerve regeneration fluid].

OBJECTIVE: To explore the protein components and the biological neutrional activity upon motor neuron, so that take a general, basic study on the nerve regeneration microenvironment which be important to facial nerve regeneration. METHOD: The animal model of rabbit facial regeneration microenvironment is established. Advanced technology of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) was used to study on the structural proteins in the regeneration fluid, as well as the motor neuron culture was used to test the biological activity in it. RESULT: 850 +/- 78 protein spots were detected in the regeneration fluid. The body area, neurite length and OD value of experimental group are higher than that of control group. CONCLUSION: We got the protein image of facial nerve regeneration fluid by 2-D PAGE, and proved that many kinds and number of proteins in the regeneration fluid have neutrional activity on motor neuron.

Animals↗

[The expression and distribution of ciliary neurotrophic factor in laryngeal nerve regeneration].

OBJECTIVE: To explore the expression and distribution of ciliary neurotrophic factor(CNTF) mRNA and its protein in laryngeal nerve regeneration. METHODS: The recurrent laryngeal nerves were sectioned and then sutured in twelve dogs. Both proximal and distal stumps of sutured region were sectioned on different postoperative days and the sections were separately used for CNTF immunohistochemistry and CNTF mRNA in situ hybridization. The area and intensity of reactive product were measured by computer image processing system. RESULTS: Strongly reactive product of CNTF mRNA and its protein deposited in myelin-related Schwann cells in normal laryngeal nerves. At week 2 following neurorrhaphy, there was very little hybridization signal and detectable CNTF immunoreactivity in distal stump and no regenerated nerve fibbers were found. At week 3, reactive product of CNTF mRNA and its protein was detected in thin Schwann cell processes ensheathing regenerated axons, while reactive product was not found in the proliferating Schwann cells which didn't ensheathe axons. CNTF immunoreactivity was also detected in the regenerated nerve axons. After long survival times, hybridization signal and the CNTF immunoreactivity in Schwamm cells became more widespread, and the area and intensity of reactive product significantly increased, but even at the longest survival time, they were still significantly less than those in intact nerve. The same change of CNTF mRNA and its protein was observed in a short segment proximal to the sutured region. CONCLUSION: CNTF expression could depend on Schwann cell-axon regeneration. The level of CNTF expression stands in striking contrast to the up-regulation of nerve growth factor in peripheral nerve degeneration and regeneration. These suggest that the exogenous CNTF might provide a supportive environment for axonal regeneration.

Animals↗

[Regeneration of the axotomised sciatic nerve in dogs using the tubulisation technique with Chitosan biomaterial preloaded with progesterone].

INTRODUCTION: Injuries to peripheral nerves can have different causes and may lead to disorders affecting mobility, sensitivity and loss of motor function as they progress. Weiss, in 1944, introduced tubulisation to promote the regeneration of a sectioned nerve. In this study the biomaterial Chitosan was used to induce and stimulate the regeneration of the sciatic nerve in dogs. At the same time, we took advantage of the characteristics offered by Chitosan to include the neurosteroid progesterone in its matrix, as a promoter of axonal growth. AIMS. The aim of our study was to determine the degree of regeneration of the sciatic nerve in dogs when axotomised tubulised with a Chitosan prosthesis steeped in the neurosteroid progesterone. MATERIALS AND METHODS: Young adult female dogs were used to evaluate the regeneration of the sciatic nerve induced at a standard of 15 mm; regeneration was determined by means of an axonal growth chamber. Nerve growth was studied through histological analysis and by electron microscope. RESULTS: The statistical analysis showed that there were no significant differences in the number of myelinated fibres between the experimental groups. The electron microscope images of the transmission in the regenerated nerves in the groups that were tubulised with Chitosan, with and without neurosteroid preloading, revealed an advanced regenerative process. This was evidenced by the fact that collagen fibres, elastin, Schwann cells and both myelinated and non myelinated fibres were observed in all cases. CONCLUSIONS: The regeneration of axotomised, tubulised nerves was achieved regardless of the treatment that was applied. The distal nerve segment that was analysed revealed a similar structure to that of a normal nerve.

Animals↗

The developmental loss of the ability of Purkinje cells to regenerate their axons occurs in the absence of myelin: an in vitro model to prevent myelination.

Axonal regeneration in the mammalian CNS is a property of immature neurons that is lost during development. Using organotypic culture of cerebellum, we have shown that in vitro Purkinje cells lose their regenerative capacity in parallel with the process of myelination. We have investigated whether myelination is involved in the age-dependent loss of regeneration of these neurons. By applying a high dose of bromodeoxyuridine in the culture medium of newborn cerebellar slices during the first 3 d in vitro, we have succeeded in obtaining cultures with oligodendrocyte depletion, together with a lack of ameboid microglia and enhancement of Purkinje cell survival. These cultures, after 14 d in vitro, are completely devoid of myelin. We have compared the ability of Purkinje cells to regenerate their axons in the presence or absence of myelin. Purkinje cells in cerebellar explants taken at birth, treated with bromodeoxyuridine and axotomized after 7 d in vitro, survive better than similar neurons in untreated cultures. However, despite the lack of myelin and the enhanced survival, Purkinje cells do not regenerate, whereas they do regenerate when the axotomy is done at postnatal day 0. Thus, the Purkinje cell developmental switch from axonal regeneration to lack of regeneration does not appear to be regulated by myelin.

Age Factors↗

Ultrastructural observations on blood vessels surrounding normal and regenerating spinal cord in newt.

The ultrastructural analysis of capillaries surrounding normal and regenerating caudal spinal cords of two species of newt (Triturus vulgaris and T. cristatus) is reported. Around normal spinal cords, capillaries were generally continuous. Surrounding the regenerating spinal cords the various capillaries of the regenerating blastema appeared discontinuous with small or broad gaps along the capillary wall. This was seen even after two months of tail regeneration when the spinal cord was similar to the normal and many axons were myelinating. These morphological findings suggest that during tail regeneration in newts the blood-brain barrier is not very effective. Also extravasating mature and immature blood cells were observed inside regenerating capillaries. Therefore growing capillaries can exchange metabolites and, possibly, growth factors with the nervous tissue and the other regenerating tissues.

Animals↗

Antigenic determinants of T lymphocyte alpha beta receptor and other leukocyte surface proteins as differential markers of skeletal muscle regeneration: detection of spatially and timely restricted patterns by MAM microscopy.

Different stages of human muscle regeneration have been identified by multiple antigen-mapping (MAM) microscopy at the level of a novel marker system. This immunofluorescence method allows the selective imaging of numerous antigen signals from cells or tissue sections. It is shown that 2 monoclonal antibodies (mAbs) against the common region of the alpha beta T lymphocyte antigen receptor (alpha beta and alpha TCR chains) and 3 mAbs against the leukocyte surface antigens Leu8, OKM5 and Leu19 recognize regenerating muscle cells at different time points of regeneration in human muscle sections. Paradoxically, these epitopes are not expressed by T lymphocytes or other mononuclear leukocytes invading regenerating muscle. Hence, presence of the corresponding antigens in muscle fibers may exclude their expression by muscle-invasive immune cells suggesting a function in muscle-specific cell-to-cell recognition. Simultaneous localization of these epitopes in Duchenne dystrophy reveals 10 different phenotypes of regenerating and normal infantile muscle cells due to different developmental stages during the myocyte differentiation. In adult muscle (mitochondrial myopathy) segmental muscle fiber necrosis is accompanied by high concentration of alpha beta/alpha TCR epitopes in the intact fiber ends, which are the target sites of myogenesis. The same sites are invaded by OKM5+ endomysial capillary sprouts that terminate at the tip of the alpha beta/alpha TCR reactive fiber ends. These hitherto unrecognized initial events of segmental muscle regeneration seem to be followed by fragmentation of the invasive capillaries into single endothelial cells, which then switch from the OKM5+ Leu19- through the OKM5+ Leu19+ to the OKM5- Leu19+ phenotype. This cell type exhibits the typical features of Leu19+ myogenic stem cells described earlier. The findings may give rise to a concept of muscle repair, in which the alpha beta/alpha TCR-related antigen, concentrated in fiber stumps, might provide positional information for invading endothelial cells. These cells appear to be a source of myogenic stem cells for regeneration.

Adult↗

[Establishment of an efficient regeneration system for Chinese cabbage cultured in vitro and histological observation of shoot development].

An efficient plant regeneration system from explants of petiole with cotyledon was developed for Chinese cabbage (Brassica campestris ssp. chinensis Makino) in our study. High frequency of shoot regeneration was obtained in the medium containing BAP 2 mg/L, NAA 0.45 mg/L and 5-7.5 mg/L AgNO3 solution to the half of NH4+ concentration's MS basic medium, with 84.3 percent being the highest frequency of shoot regeneration in seven Chinese cabbage cultivars and one parentage, and the new formed shoots were observed six days after inoculation. Factors influencing in vitro explant regeneration were further studied. The results showed that there was positive correlation between frequency of shoot regeneration and number of shoots per explant. The regeneration plants produced flowers and fruits after about 50 days with low temperature treatment from 8 degrees C to 16 degrees C. Moreover, histological observation indicated that the mode of plant regeneration in B. campestris ssp. chinensis was similar to that of other Brassica crops, and adventitions buds were formed from globular meristematic cluster which derived from the cells of vascular parenchyma at the cut surface of petiole.

Brassica↗

The role of hepatic stimulator substance (HSS) on liver regeneration arrest induced by cadmium.

BACKGROUND: The mechanism of cadmium-induced liver regeneration arrest in relation to hepatic stimulator substance (HSS) biological activity was investigated. MATERIALS AND METHODS: In Wistar rats subjected to 65 - 70% partial hepatectomy, saline, cadmium, cadmium and HSS were administered. The rats were also subjected to 30 - 34% partial hepatectomy. Mitotic index, immunochemistry for PCNA, 3[H]-thymidine incorporation into DNA and thymidine kinase activity were used as indices of liver regeneration. HSS biological activity was evaluated in all groups of rats using a bioassay. RESULTS: Liver regeneration and HSS activity were arrested by cadmium during the first 24 h after partial hepatectomy. Both in normal and in cadmium-treated rats, the HSS activity was increased and liver regeneration coincided. HSS activity was stable in 30 - 34% hepatectomized rats. HSS administration was able to restore liver regeneration arrest induced by cadmium. CONCLUSION: The biological activity of HSS increased at the time of G1/S transition of hepatocytes in the cell cycle and no increase was observed with asynchronous G1/S transition (30 - 34% partial hepatectomy). The suppression of HSS biological activity by cadmium seems to represent an important factor for liver regeneration arrest induced by the metal and HSS administration is able to restore liver regeneration.

Animals↗

[The value of methods of bone regeneration evaluation in limb lengthening by the Wagner, Ilizarov methods and by physeal distraction].

Limb lengthening is a long-lasting process, and during new bone formation different complications may occur. Due to this, early diagnosis of disturbances of new bone formation leading to such complications is of importance. The goal of this study is to analyze already used methods of regenerate evaluation. Material consists of retrospective data of 237 patients, who underwent limb lengthening between 1983 and 2002 by one of three methods: Wagner method, Ilizarow method and physeal distraction. During femoral lengthening by Wagner method appropriate shape of regenerate according to Hamanishi was observed in 9 cases (29.0%), and during tibia lengthening--only in 1 case (6.7%). During femoral lengthening by physeal distraction appropriate shape of regenerate (A or B according to Hamanishi) was observed in 24 cases (77.4%), and during tibia lengthening--in 11 cases (78.6%). During femoral lengthening by Ilizarow method appropriate shape of regenerate was observed in 51 cases (72.9%), and during tibia lengthening--in 46 cases (66.7%). Only in Wagner method a correlation between abnormal regenerate shape and bone consolidation complications was noted. Methods of evaluation of bone regeneration during distraction osteogenesis give only descriptive assessment. So far parameters applied for evaluation of distraction osteogenesis in Ilizarow method and physeal distraction do not allow for detailed assessment of bone regeneration process.

Adolescent↗

Long-term potentiation and activity-dependent retinotopic sharpening in the regenerating retinotectal projection of goldfish: common sensitive period and sensitivity to NMDA blockers.

The regenerating retinotectal projection in goldfish goes through an activity-driven refinement that appears to involve the elimination of inappropriate branches from early arbors. Retinotopically appropriate branches may be stabilized because the normally correlated firing of neighboring ganglion cells causes summation of their postsynaptic responses and increases their effectiveness by a Hebbian mechanism. In this study, I report that the regenerating projection has an increased capacity for long-term potentiation (LTP) that may be related to the activity-driven sharpening. In the normal projection, field potentials, reflecting currents from EPSPs elicited by optic nerve shock, are large (greater than 4 mV) and very stable. In newly regenerated projections, field potentials are initially small (less than 1 mV), but a train of 20 stimuli at 0.1 Hz results in a large (100-200%) increase in amplitude that is stable for at least 8 hr, and in 3 cases overnight. The capacity for potentiation is greatest from 20 to 40 d postcrush, the time just after arrival of the optic fibers, and during the period of retinotopic sharpening. A greater-than-normal capacity for potentiation persists for many months. Topical application of NMDA receptor blockers AP5 or AP7 at 25 microM prevents potentiation without decrementing ongoing responses. The closely related agent AP6, which is not an NMDA receptor blocker, does not prevent potentiation. In addition, infusion of the NMDA receptor blockers AP5 or AP7 into the tectal ventricle (4 microliters/d of 500 microM solution) for 2-3 weeks during regeneration prevented retinotopic sharpening, as assessed by electrophysiological mapping. At each tectal point, responsive areas in the visual field were enlarged to 28 degrees vs 11-12 degrees in control regenerates and normals. This was comparable to data from fish regenerating with activity blocked with intraocular tetrodotoxin or synchronized by stroboscopic illumination and indicates uncorrected errors in targeting of regenerated arbors (Schmidt, 1985). The results support the involvement of NMDA receptors in sharpening and suggest that the initial step in stabilizing appropriate branches may be a long-lasting increase in synaptic gain.

Animals↗

Influence of malnutrition on regeneration and composition of the liver in rats.

Using a new experimental model of protein-calorie starvation, the impact of malnutrition on liver regeneration, and the changes in liver composition during malnutrition and regeneration, were studied. Forty-one rats were allocated to one of 3 groups: group I (n = 13) were normally nourished rats which underwent partial hepatectomy, group II (n = 16) were semistarved rats which underwent partial hepatectomy, and group III (n = 12) were normally nourished rats which underwent sham operations. Liver biopsy specimens were taken at the time of partial hepatectomy and when the rats were killed after 48 hours of regeneration. The samples were used for determination of water, fat, glycogen, protein, DNA, and RNA content. The replication rate of liver cells was measured by autoradiography after continuous incorporation of tritium labelled thymidine. Malnutrition was associated with a higher postoperative mortality and a reduced rate of regeneration. The livers changed during malnutrition with a reduction in both size and glycogen concentration. After 48 hours of regeneration there was a marked change in liver composition with severe fatty degeneration, a fall in glycogen and a rise in water content. RNA concentration was stimulated during regeneration, but during malnutrition the protein content decreased. The results emphasise the importance of an adequate nutritional state for liver regeneration.

Animals↗