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PubMed2005

Ontogenetic decline of regenerative ability and the stimulation of human regeneration.

Although we cannot regenerate our limbs today, it is likely that when we were embryos we could regenerate many of our tissues, including our limbs. Like other vertebrates, our impressive regenerative abilities were lost during embryogenesis, leaving us with a relatively limited ability to repair tissue damage. In contrast, adult salamanders can reactivate the embryonic regeneration response, and thus they provide the opportunity to discover the principles and mechanisms of tissue and organ regeneration. One important lesson we have learned from salamanders is that regeneration occurs in two steps. While the second step shares the mechanisms of growth control and pattern formation with limb development, the first step is unique and leads to the formation of a regeneration blastema. A second lesson is that connective tissue fibroblasts control regeneration, and that the unique regenerative ability of salamanders (the first step of regeneration) is a consequence of the ability of fibroblasts to dedifferentiate and give rise to blastema cells. Since we all developed limbs as embryos, we all possess the genetic program for making a limb (the second step of regeneration). Therefore, the challenge for inducing limb regeneration in humans is to discover how to induce fibroblast dedifferentiation.

Animals↗
PubMed1987

Maturation, dystrophic changes and the continuous production of fibers in skeletal muscle regenerating in the absence of nerve.

To investigate how much skeletal muscle depends on influences coming from peripheral nerve, we studied muscle regeneration induced by bupivacaine in the permanently denervated soleus muscle of adult rats. Using light and electron microscopy, we studied the extent of maturation attained by regenerated myofibers and their ability to regenerate again after repeated bupivacaine injury. Morphometric analyses showed that within the first two weeks after injury, the regenerated denervated fibers increased in diameter, matured and then became atrophic. By electron microscopy the morphological characteristics of mature fibers are evident, but final differentiation into adult fiber types is not attained. This is in keeping with previously reported biochemical results. A new phase of massive regeneration occurred when bupivacaine treatment was repeated, thus demonstrating that regenerated myofibers devoid of innervation keep their intrinsic ability to regenerate after injury. Spontaneous though scanty regeneration also occurred in denervated regenerated muscle at late stages, when atrophy and dystrophy of fibers became prominent. Concomitantly the relative number of myosatellite cells remained high. These morphological observations further support the hypothesis that fiber regeneration participates in the maintenance of chronically denervated muscles.

Animals↗
PubMed2000

Rabbit facial nerve regeneration in autologous nerve grafts after antecedent injury.

OBJECTIVE: The effect of incomplete antecedent injuries on subsequent facial nerve regeneration within cable graft repairs is not known. The purpose of this study is to compare facial nerve regeneration after an immediate and delayed neural cable graft repair. METHOD: Rabbit facial nerve regeneration after complete transectional injuries of the buccal division was compared in two experimental models. In one, a 10-mm segment of the nerve was transected, rotated 180 degrees, and immediately repaired as a cable graft (N=8). In the second, a preliminary nerve crush was allowed to recover over a 4-week period and a 10-mm segment of nerve centered on the crush site was then transected, rotated 180 degrees, and delay repaired as a cable graft (N = 7). Data are presented as total numbers of regenerating myelinated axons that traverse the surgical repair to innervate the cable graft and distal nerve stumps, as well as the percentage of regenerating neurites compared with preoperative pooled and individual controls. Subpopulations of regenerating neurons are delineated to quantify the pattern of neural innervation. RESULTS: Five weeks after cable graft repair both groups had similar myelinated outgrowth from the proximal nerve stump across the proximal anastomosis to innervate the cable graft (3995 +/- 1209 vs. 3284 +/- 651; P = .89). However, the delayed repair group had more intrafascicular regeneration within cable grafts (2261 +/- 931 vs. 1660 +/- 1169; P = .02) and distal nerve stump (1532 +/- 281 vs. 445 +/- 120; P = .004) than the immediate repair group. The immediate repair group had greater extrafascicular nerve regeneration in the cable graft (2335 +/- 1954 vs. 437 +/- 236; P = .001) and more myelin and axonal debris in pre-existing neural fascicles of the cable graft (P = .02) and distal nerve stump (463 +/- 187 vs. 103 +/- 87; P = .02). CONCLUSIONS: Antecedent priming lesions do not enhance axonal survival as determined by regenerating myelinated axonal counts. However, antecedent injuries enhance the efficiency of neural innervation of the affected mimetic musculature by increasing the number of myelinated intrafascicular neural regenerants in the cable graft and distal nerve stump. This is accomplished by two factors: increased perineural fibrosis and decreased intrafascicular myelin and axonal debris.

Animals↗
PubMed2006

Transcriptional profiling of caudal fin regeneration in zebrafish.

Regeneration of severed limbs in adult animals is restricted to urodele amphibians. Mammals, including humans, have very limited regenerative capabilities and even with proper treatment, only the tips of our digits can grow back. Teleost fish can regenerate amputated fins, the evolutionary ancestors of limbs. To elucidate the principles of limb-fin regeneration, we performed an Affymetrix microarray screen on regenerating caudal fins 12, 24, 48, and 72 h post amputation. Approximately 15,000 zebrafish transcripts were analyzed, identifying 829 transcripts as differentially expressed during regeneration. Of those, 563 were up-regulated and 266 were down-regulated. We constructed a comprehensive database containing expression data, functional assignment, and background information from the literature for each differentially expressed transcript. In order to validate our findings, we employed three approaches: (1) microarray expression analysis of genes previously implicated in fin regeneration, (2) RT-PCR analysis of genes newly identified as differentially expressed during regeneration, and (3) in situ hybridization of the up-regulated genes bambi, dlx5A, and her6. Moreover, we show that Smad 1/5/8 proteins, effector molecules of Bmp signaling, are phosphorylated during fin regeneration. Taken together, we provide a comprehensive database of fin regeneration that will serve as an important tool for understanding the molecular mechanisms of regeneration.

Animals↗
PubMed2006

Two different transgenes to study gene silencing and re-expression during zebrafish caudal fin and retinal regeneration.

We used the 500-bp Xenopus ef1-alpha promoter and the 2-kb zebrafish histone 2A.F/Z promoter to generate several independent transgenic zebrafish lines expressing EGFP. While both promoters drive ubiquitous EGFP expression in early zebrafish development, they are systematically silenced in several adult tissues, including the retina and caudal fin. However, EGFP expression is temporarily renewed in the adult during either caudal fin or retinal regeneration. In the Tg(H2A.F/Z:EGFP)nt line, EGFP is moderately expressed in both the wound epithelium and blastema of the regenerating caudal fin. In the Tg(ef1-alpha:EGFP)nt line, EGFP expression is reinitiated and restricted to the blastema of the regenerating caudal fin and colabels with BrdU, PCNA, and msxc-positive cells. Thus, these two ubiquitous promoters drive EGFP transgene expression in different cell populations during caudal fin regeneration. We further analyzed the ability of the ef1-alpha:EGFP transgene to label nonterminally differentiated cells during adult tissue regeneration. First, we demonstrated that the transgene is highly methylated in adult zebrafish caudal fin tissue, but not during fin regeneration, implicating methylation as a potential means of transgene silencing in this line. Next, we determined that the ef1-alpha:EGFP transgene is also re-expressed during adult retinal regeneration. Specifically, the ef1-alpha:EGFP transgene colabels with PCNA in the Müller glia, a specialized cell that is the source of neuronal progenitors during zebrafish retinal regeneration. Thus, we concluded that Tg(ef1-alpha:EGFP)nt line visually marks nonterminally differentiated cells in multiple adult regeneration environments and may prove to be a useful marker in tissue regeneration studies in zebrafish.

Animals↗
PubMed2005

Initial upregulation of growth factors and inflammatory mediators during nerve regeneration in the presence of cell adhesive peptide-incorporated collagen tubes.

Neurotrophic factors play an important modulatory role in axonal sprouting during nerve regeneration involving the proliferation of hematogenous and Schwann cells in damaged tissue. We have exposed lesioned sciatic nerves to a collagen prosthesis with covalently bonded small cell adhesive peptides Arg-Gly-Asp-Ser (RGDS), Lys-Arg-Asp-Ser (KRDS), and Gly-His-Lys (GHK) to study local production of growth factors and cytokines in the regenerating tissues. Western/enzyme-linked immunosorbent assay (ELISA) studies were performed after 10 days of regeneration, when the tubular prosthesis is filled with fibrous matrix infiltrated by hematogenous cells and proliferating Schwann cells with growth factors produced locally. Regeneration was also analyzed by morphometrical methods after 30 days. The quantification of growth factors and proteins by ELISA revealed that there was an enhanced expression of the neurotrophic factors nerve growth factor (NGF) and neurotrophins (NT-3 and NT-4) in the regenerating tissues. This was further established by Western blot to qualitatively analyze the presence of the antigens in the regenerating tissues. Schwann cells were localized in the regenerating tissues using antibodies against S-100 protein. Other growth factors including growth-associated protein 43 (GAP-43), apolipoprotein E (Apo E), and pro-inflammatory cytokine like interleukin-1alpha (IL-1alpha) expression in the peptide groups were evaluated by ELISA and confirmed by Western blotting. Cell adhesive integrins in the proliferating cells were localized using integrin-alpha V. The combined results suggest that the early phase of regeneration of peripheral nerves in the presence of peptide-incorporated collagen tubes results in the enhanced production of trophic factors by the recruited hematogenous cells and Schwann cells, which in turn help in the secretion of certain vital trophic and tropic factors essential for early regeneration. Furthermore, hematogenous cells recruited within the first 10 days of regeneration help in the production of inflammatory mediators like interleukins that in turn stimulate Schwann cells to produce NGF for axonal growth.

Absorbable Implants↗
PubMed2005

Restoration of hepatic mast cells and expression of a different mast cell protease phenotype in regenerating rat liver after 70%-hepatectomy.

Mast cells (MC) can undergo significant changes in number and phenotype; these alterations result in the differential expression of growth factors and cytokines. Kit ligand (KL; stem cell factor) is produced by mesenchymal cells, and in the liver by biliary epithelial cells. Recent studies suggest that KL, and its receptor c-kit, may be involved in liver regeneration after loss of liver mass. However, KL is also the major growth, differentiating, chemotactic, and activating factor for MC. The aim of our study was to elucidate the dynamics and phenotype of hepatic MC and KL/c-kit expression during liver regeneration after partial (70%) hepatectomy in the rat. Regenerating livers were harvested after 1, 3, 7, and 14 days, respectively (n = 6 each day). MC were stained for naphthol-AS/D-chloroacetate esterase and counted as MC per bile ductule. MC phenotype was assessed by rat MC protease (RMCP)-1 and -2 immunofluorescence staining, in order to distinguish RMCP-1 positive connective tissue MC (CTMC) from RMCP-2 positive mucosa MC (MMC). mRNA expression of RMCP, c-kit, and the differentially spliced variants of KL was quantified by RT-PCR. MC counts per bile ductule decreased in regenerating rat liver tissue at day 3, compared with native livers, and became normal thereafter. Hepatic MC were predominantly of a CTMC phenotype expressing RMCP-1, as previously published; after hepatectomy, between 76 and 99% of all MC double-expressed RMCP-1 and -2, compatible with an MMC phenotype. The ratio of the two alternatively spliced mRNAs for KL (KL-1 : KL-2), and c-kit mRNA expression did not differ significantly between regenerating livers and the livers of sham operated animals. These results suggest that hepatic mast cells are restored during liver regeneration after partial hepatectomy in the rat. Restored MC express an MMC phenotype, suggesting migration from outside into the regenerating liver. Alternative splicing of KL is affected by the surgical procedure in general, and, together with its receptor c-kit, doesn't seem to be involved in liver regeneration after partial hepatectomy in the rat. Further functional studies, and studies in regenerating human livers might offer the possibility of elucidating the role of the hepatic mast cell, and its different protease phenotypes during liver regeneration after surgical loss of liver mass.

Animals↗
PubMed2006

Mesenchymal stem cells and bone regeneration.

OBJECTIVE: To review the role of mesenchymal stem cells (MSC) in bone formation and regeneration, and outline the development of strategies that use MSC in bone healing and regeneration. STUDY DESIGN: Literature review. METHODS: Medline review, synopses of authors' published research. RESULTS: The MSC is the basic cellular unit of embryologic bone formation. Secondary bone healing mimics bone formation with proliferation of MSC then their differentiation into components of fracture callus. Bone regeneration, where large amounts of bone must form, mimics bone healing and can be achieved with MSC combined with strategies of osteogenesis, osteoinduction, osteoconduction, and osteopromotion. MSC based strategies first employed isolated and culture expanded stem cells in an osteoconductive carrier to successfully regenerate a critical segmental defect in the femur of dogs, which was as effective as autogenous cancellous bone. Because MSC appeared to be immunologically privileged, a study using mismatched allogeneic stem cells demonstrated that these cells would regenerate bone without inciting an immunologic response, documenting the possibility of banked allogeneic MSC for bone regeneration. A technique was developed for selectively retaining MSC from large bone marrow aspirates at surgery for bone regeneration. These techniques utilized osteoconductive and osteoinductive carriers and resulted in bone regeneration that was similar to autogenous cancellous bone. CONCLUSION: MSC can be manipulated and combined with carriers that will result in bone regeneration of critically sized bone defects. CLINICAL RELEVANCE: These techniques can be employed clinically to regenerate bone and serve as an alternative to autogenous cancellous bone.

Animals↗
PubMed1985

Intracellular analysis of the innervation of a crayfish sensory interneuron by regenerating afferents.

Mechanoreceptors of the crayfish tail fan have peripheral somata and send their axons to the last (sixth) abdominal ganglion via five bilateral pairs of nerve roots (R1-R5). Comparisons were made between normal crayfish and regenerate preparations in which R4 had been cut and directed back to an extensively denervated sixth abdominal ganglion; 8 to 15 weeks postoperatively, an identified target interneuron (A) in this ganglion was impaled, and its response to water currents, electrical excitation of R4, and stimulation of individual sensory hairs supplying axons to R4 was studied along with several other properties of the pre- and post-synaptic neurons. Normal levels of excitability in A to R4 stimulation were achieved within six weeks as judged by extracellular criteria. Subsequent intracellular analysis revealed that few differences exist between regenerated and normal inputs: probability of (re-) connection, unitary EPSP amplitude and time course distributions, resting membrane potentials, and critical firing levels were comparable in the two groups; input impedance, however, may have been lower in regenerates. Compound electrically elicited EPSPs were similar in amplitude, rise time, and half amplitude width, but differed slightly in latency to onset (regenerates greater than normals). This was accounted for by differences in conduction time to the ganglion in regenerates, and central delay estimates suggest that connections in both groups are monosynaptic. The body root (R1) providing input to A that remained intact in the regenerate preparations increased in efficacy; over 12 postoperative weeks the response of A to R1 activation by water drops steadily increased and at 12 weeks unitary increments to ascending electrical stimulation of R1 were significantly larger than in normals. The response to giant interneuron activation demonstrated that recurrent inhibitory inputs were normal in regenerates. In addition, synaptic depression, normally responsible for behavioral habituation in this system, was comparable across groups. Further, protection from habituation was observed in both normals and regenerates if R4 stimulation was preceded by giant interneuron activation, thus indicating that normal presynaptic inhibitory inputs to the regenerated afferent terminals have also successfully regenerated.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed2003

Digit regeneration is regulated by Msx1 and BMP4 in fetal mice.

The regeneration of digit tips in mammals, including humans and rodents, represents a model for organ regeneration in higher vertebrates. We had previously characterized digit tip regeneration during fetal and neonatal stages of digit formation in the mouse and found that regenerative capability correlated with the expression domain of the Msx1 gene. Using the stage 11 (E14.5) digit, we now show that digit tip regeneration occurs in organ culture and that Msx1, but not Msx2, mutant mice display a regeneration defect. Associated with this phenotype, we find that Bmp4 expression is downregulated in the Msx1 mutant digit and that mutant digit regeneration can be rescued in a dose-dependent manner by treatment with exogenous BMP4. Studies with the BMP-binding protein noggin show that wild-type digit regeneration is inhibited without inhibiting the expression of Msx1, Msx2 or Bmp4. These data identify a signaling pathway essential for digit regeneration, in which Msx1 functions to regulate BMP4 production. We also provide evidence that endogenous Bmp4 expression is regulated by the combined activity of Msx1 and Msx2 in the forming digit tip; however, we discovered a compensatory Msx2 response that involves an expansion into the wild-type Msx1 domain. Thus, although both Msx1 and Msx2 function to regulate Bmp4 expression in the digit tip, the data are not consistent with a model in which Msx1 and Msx2 serve completely redundant functions in the regeneration response. These studies provide the first functional analysis of mammalian fetal digit regeneration and identify a new function for Msx1 and BMP4 as regulators of the regenerative response.

Animals↗
PubMed2004

The hedgehog pathway is a modulator of retina regeneration.

The embryonic chick has the ability to regenerate its retina after it has been completely removed. Here, we provide a detailed characterization of retina regeneration in the embryonic chick at the cellular level. Retina regeneration can occur in two distinct manners. The first is via transdifferentiation, which is induced by members of the Fibroblast growth factor (Fgf) family. The second type of retinal regeneration occurs from the anterior margin of the eye, near the ciliary body (CB) and ciliary marginal zone (CMZ). We show that regeneration from the CB/CMZ is the result of proliferating stem/progenitor cells. This type of regeneration is also stimulated by Fgf2, but we show that it can be activated by Sonic hedgehog (Shh) overexpression when no ectopic Fgf2 is present. Shh-stimulated activation of CB/CMZ regeneration is inhibited by the Fgf receptor (Fgfr) antagonist, PD173074. This indicates that Shh-induced regeneration acts through the Fgf signaling pathway. In addition, we show that the hedgehog (Hh) pathway plays a role in maintenance of the retina pigmented epithelium (RPE), as ectopic Shh expression inhibits transdifferentiation and Hh inhibition increases the transdifferentiation domain. Ectopic Shh expression in the regenerating retina also results in a decrease in the number of ganglion cells present and an increase in apoptosis mostly in the presumptive ganglion cell layer (GCL). However, Hh inhibition increases the number of ganglion cells but does not have an effect on cell death. Taken together, our results suggest that the hedgehog pathway is an important modulator of retina regeneration.

Animals↗
PubMed1993

Analysis of Hox-4.5 and Hox-3.6 expression during newt limb regeneration: differential regulation of paralogous Hox genes suggest different roles for members of different Hox clusters.

Adult urodele amphibians can regenerate their limbs and tail. Based on their roles in other developing systems, Hox genes are strong candidates for genes that play a role in regulating pattern formation during regeneration. There are four homologous clusters of Hox genes in vertebrate genomes. We isolated cDNA clones of two newt homeobox genes from homologous positions within two Hox clusters; Hox-4.5 and Hox-3.6. We used RNase protection on nonamputated (normal) and regenerating newt appendages and tissue to compare their transcriptional patterns. Both genes show increased expression upon amputation with similar kinetics. Hox-4.5 and Hox-3.6 transcription is limited to the mesenchymal cells in the regenerates and is not found in the epithelial tissue. In addition to regenerating appendages, both genes are transcriptionally active in adult kidney of the newt. Striking differences were found in the regulation of Hox-4.5 and Hox-3.6 when they were compared in unamputated limbs and in regenerating forelimbs versus regenerating hindlimbs. Hox-4.5 is expressed in the blastema of regenerating fore- and hindlimbs, but Hox-4.5 transcripts are not detectable in normal limbs. In contrast, Hox-3.6 transcripts are found exclusively in posterior appendages, but are present in normal as well as regenerating hindlimbs and tails. Hox-4.5 is also expressed at a higher level in proximal (mid-humerus) regenerates than in distal ones (mid-radius). When we proximalized the positional memory of a distal blastema with retinoic acid, we find that the early expression level of Hox-4.5 is also proximalized. When the expression of these genes is compared to the expression of two previously reported newt Hox genes, a consistent pattern emerges, which can be interpreted in terms of differential roles for the different Hox clusters in determining regenerative limb morphology.

Amino Acid Sequence↗
PubMed2000

Zebrafish kit mutation reveals primary and secondary regulation of melanocyte development during fin stripe regeneration.

Fin regeneration in adult zebrafish is accompanied by re-establishment of the pigment stripes. To understand the mechanisms underlying fin stripe regeneration and regulation of normal melanocyte stripe morphology, we investigated the origins of melanocytes in the regenerating fin and their requirement for the kit receptor tyrosine kinase. Using pre-existing melanin as a lineage tracer, we show that most fin regeneration melanocytes develop from undifferentiated precursors, rather than from differentiated melanocytes. Mutational analysis reveals two distinct classes of regeneration melanocytes. First, an early regeneration class develops dependent on kit function. In the absence of kit function and kit-dependent melanocytes, a second class of melanocytes develops at later stages of regeneration. This late kit-independent class of regeneration melanocytes has little or no role in wild-type fin stripe development, thus revealing a secondary mode for regulation of fin stripes. Expression of melanocyte markers in regenerating kit mutant fins suggests that kit normally acts after mitf and before dct to promote development of the primary kit-dependent melanocytes. kit-dependent and kit-independent melanocytes are also present during fin stripe ontogeny in patterns similar to those observed during regeneration.

Animals↗
PubMed1989

Regeneration of the eighth cranial nerve. III. Central projections of the primary afferent fibers from individual vestibular receptors in the bullfrog.

The projection site in the central nervous system of individual end-organ branches of regenerated vestibular nerves was documented in bullfrogs. The eighth cranial nerve was surgically sectioned in 12 bullfrogs (Rana catesbeiana) and allowed to regenerate. Horseradish peroxidase was used to label the primary vestibular afferent fibers from the whole nerve (one frog) and the whole anterior branch (two frogs), and the individual receptor end-organ branches to the anterior semicircular canal (three frogs), the horizontal semicircular canal (three frogs), and the sacculus (three frogs). A sufficient number of fibers from the individual receptors were labeled in nine specimens to allow a description of their central projection pattern. The overall pattern of central projections of the regenerated nerves was similar to the pattern of normal nerves. In the eighth nerve root proper and in the area proximal to the vestibular nuclei, fibers from the anterior semicircular canal were ventral, fibers from the sacculus and posterior branch were dorsal, and fibers from the horizontal semicircular canal were in an intermediate position. In ventral, superior, and descending vestibular nuclei, a projection pattern was also identifiable in some specimens. The fibers of the anterior semicircular canal projected ventral to the fibers of the horizontal semicircular canal, with fibers from the posterior branch more dorsal. The saccular fibers projected to the dorsal aspect of the ventral nucleus and the ventral aspect of the dorsal nucleus. For the most part, projections in the regenerated specimens, as verified histologically, were comparable to individual end-organ projections previously identified in normal frogs. The only major discrepancy between the findings from the regenerated specimens and those from normal frogs was in the position of the vestibular tract and the innervation to the medial nucleus. The regenerated vestibular tract was shifted laterally in the brain stem. Regenerated thick and thin fibers were intermixed in the tract and projected to the medial nucleus. Normally, the thin fibers are lateral to the thick fibers in the tract. Therefore, the regenerated afferent fibers of the vestibular nerve selectively reinnervated the vestibular nuclei, and the fibers to the individual end-organs selectively reinnervated particular parts of the nuclei. In addition, the thick and thin regenerated fibers preserved the type of bouton endings normally found. The regenerated thick fibers had rare boutons en passant and a great number of collaterals, while the thin fibers had numerous boutons en passant and only a few collaterals.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2005

Stability and repeat regeneration potential of the engineered liver tissues under the kidney capsule in mice.

Liver tissue engineering using hepatocyte transplantation has been proposed as a therapeutic alternative to liver transplantation toward several liver diseases. We have previously reported that stable liver tissue with the potential for liver regeneration can be engineered at extrahepatic sites by transplanting mature hepatocytes into an extracellular matrix. The present study was aimed at assessing the liver tissue persistence after induced regeneration by hepatectomy and repeat regeneration potential induced by repeat hepatectomy. Mouse isolated hepatocytes mixed in EHS extracellular matrix gel were transplanted under both kidney capsules of isogenic mice. The hepatocyte survival persisted for over 25 weeks. In some of the mice, we confirmed that the grafted hepatocytes developed a thin layer of liver tissues under the kidney capsule, determined by specific characteristics of differentiated hepatocytes in cord structures between the capillaries. We then assessed the regenerative potential and persistence of the exogenous liver tissue. To induce liver regeneration, we performed a two-thirds hepatectomy at 70 days after hepatocyte transplantation. Three weeks after this procedure, the engineered liver tissues showed active regeneration, reaching serum marker protein levels of 261 +/- 42% of the prehepatectomy level. We found that the regenerated liver tissue was stably maintained for 100 days (length of the experiment). Repeat regeneration potential was established by performing a repeat hepatectomy (that had been two-thirds hepatectomized at day 70) 60 days after the initial hepatectomy. Again, the regenerated engineered liver tissues showed active regeneration as there was an approximately twofold increase in the serum marker protein levels. The present studies demonstrate that liver tissue, which was recognized as a part of the host naive liver in terms of the regeneration profile, could be engineered at a heterologous site that does not have access to the portal circulation.

Animals↗
PubMed2002

Bcl-2 overexpression does not enhance in vivo axonal regeneration of retinal ganglion cells after peripheral nerve transplantation in adult mice.

Optic nerve (ON) injury in adult mammals causes retinal ganglion cell (RGC) death and subsequent visual loss. Recovery of vision requires both rescuing axotomized RGCs and inducing their axonal regeneration. Axotomized RGCs are significantly rescued by overexpression of bcl-2, an anti-apoptotic gene. However, whether bcl-2 affects axonal regeneration is controversial. In neonatal bcl-2 transgenic mice (bcl-2 mice), optic tract regeneration after tectal lesion was promoted (Chen et al., 1997), whereas ON regeneration after ON crush was not (Lodovichi et al., 2001). These conflicting results may be attributable to different environments between tectum and ON. We tested here whether bcl-2 overexpression enhances in vivo RGC axonal regeneration in adult mice through a permissive environment in the peripheral nerve (PN) graft. Four weeks after PN transplantation to the proximal ON stump, we assessed the number of surviving and regenerating RGCs by retrograde labeling. Although the survival rate in bcl-2 mice was significantly enhanced compared with that in wild-type (wt) mice, the regeneration rate was not enhanced. In both bcl-2 and wt mice, RT97 immunostaining of the PN-grafted retinas revealed some RGC axons regrowing intraretinally but repulsed at the optic disk. To circumvent this repulsive barrier, we directly transplanted the PN graft to the partially injured retina and compared regeneration rates between these mice. Here again the regeneration rate in bcl-2 mice did not exceed that in wt mice. These findings indicate that bcl-2 overexpression enhances survival but not axonal regeneration of adult RGCs even within a permissive environment.

Animals↗
PubMed2000

Cell mosaic patterns in the native and regenerated inner retina of zebrafish: implications for retinal assembly.

In part because of its laminar organization and morphologically distinct cell populations, the vertebrate retina has often been used as a system for investigating the assembly of neural structures. The retinas of adult teleost fish, because they grow throughout life and can regenerate following an injury, provide an especially attractive model system for such investigations. In an effort to provide a quantitative foundation for testing hypotheses regarding the mechanisms of pattern formation during growth and regeneration of the vertebrate retina, nearest neighbor and auto-correlation analyses were used to examine the mosaic patterns of eight inner retinal cell groups in the native and regenerated retina of adult zebrafish. In both native and regenerated retina, the mosaic patterns of most inner retinal cells are non-random. However, regenerated mosaics tend toward significantly lower nearest neighbor distances, less orderly patterns, and more variable radial locations than their native retina counterparts. The individual cell groups in both native and regenerated inner retina are likely to be spatially distributed independently. The results support the hypotheses that, in the adult zebrafish: 1) distinct inner retinal cell groups of native retina are also present in regenerated retina; 2) the assembly of inner retinal cell mosaics is controlled by non-random spatial organizing mechanisms during development, growth, and regeneration; and 3) the spatial organization of cell mosaics is disrupted during regeneration. The results suggest that retinal regeneration may represent a spatially disrupted recapitulation of retinal developmental mechanisms.

Animals↗
PubMed2000

The transitional zone and CNS regeneration.

Most nerves are attached to the neuraxis by rootlets. The CNS-PNS transitional zone (TZ) is that length of rootlet containing both central and peripheral nervous tissue. The 2 tissues are separated by a very irregular but clearly defined interface, consisting of the surface of the astrocytic tissue comprising the central component of the TZ. Central to this, myelin sheaths are formed by oligodendrocytes and the supporting tissue is astrocytic. Peripheral to it, sheaths are formed by Schwann cells which are enveloped in endoneurium. The features of transitional nodes are a composite of those of central and peripheral type. The interface is penetrated only by axons. It is absent at first. It is formed by growth of processes into the axon bundle from glial cell bodies around its perimeter. These form a barrier across the bundle which fully segregates prospectively myelinated axons. Rat spinal dorsal root TZs have been used extensively to study CNS axon regeneration. The CNS part of the TZ responds to primary afferent axon degeneration and to regenerating axons in ways which constitute a satisfactory model of the gliotic tissue response which occurs in CNS lesions. It undergoes gliosis and the gliotic TZ tissue expands distally along the root. In mature animals axons can regenerate satisfactorily through the endoneurial tubes of the root but cease growth on reaching the gliotic tissue. The general objective of experimental studies is to achieve axon regeneration from the PNS through this outgrowth and into the dorsal spinal cord. Since immature tissue has a greater capacity for regeneration than that of the adult, one approach includes the transplantation of embryonic or fetal dorsal root ganglia into the locus of an extirpated adult ganglion. Axons grow centrally from the transplanted ganglion cells and some enter the cord. Other approaches include alteration of the TZ environment to facilitate axon regeneration, for example, by the application of tropic, trophic, or other molecular factors, and also by transplantation of cultured olfactory ensheathing cells (OECs) into the TZ region. OECs, by association with growing axons, facilitate their extensive regeneration into the cord. Unusually, ventral motoneuron axons may undergo some degree of unaided CNS regeneration. When interrupted in the spinal cord white matter, some grow out to the ventral rootlet TZ and thence distally in the PNS. The DRTZ is especially useful for quantitative studies on regeneration. Since the tissue is anisometric, individual parameters such as axon numbers, axon size and glial ensheathment can be readily measured and compared in the CNS and PNS environments, thereby yielding indices of regeneration across the interface for different sets of experimental conditions.

Animals↗