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The process of regeneration is most readily studied in species of sponge, hydra, planarian and salamander (i.e., newt and axolotl). The closure of MRL mouse ear pinna through-and-through holes provides a mammalian model of unusual wound healing/regeneration in which a blastema-like structure closes the ear hole and cartilage and hair follicles are replaced. Recent studies, based on a broad level of DNA damage and a cell cycle pattern of G₂/M "arrest," showed that p21(Cip1/Waf1) was missing from the MRL mouse ear and that a p21-null mouse could close its ear holes. Given the p53/p21 axis of control of DNA damage, cell cycle arrest, apoptosis and senescence, we tested the role of p53 in the ear hole regenerative response. Using backcross mice, we found that loss of p53 in MRL mice did not show reduced healing. Furthermore, cross sections of MRL. p53(-/-) mouse ears at 6 weeks post-injury showed an increased level of adipocytes and chondrocytes in the region of healing whereas MRL or p21(-/-) mice showed chondrogenesis alone in this same region, though at later time points. In addition, we also investigated other cell cycle-related mutant mice to determine how p21 was being regulated. We demonstrate that p16 and Gadd45 null mice show little healing capacity. Interestingly, a partial healing phenotype in mice with a dual Tgfβ/Rag2 knockout mutation was seen. These data demonstrate an independence of p53 signaling for mouse appendage regeneration and suggest that the role of p21 in this process is possibly through the abrogation of the Tgfβ/Smad pathway.

Animals capable of regenerating multiple tissue types, organs, and appendages after injury are common yet sporadic and include some sponge, hydra, planarian, and salamander (i.e., newt and axolotl) species, but notably such regenerative capacity is rare in mammals. The adult MRL mouse strain is a rare exception to the rule that mammals do not regenerate appendage tissue. Certain commonalities, such as blastema formation and basement membrane breakdown at the wound site, suggest that MRL mice may share other features with classical regenerators. As reported here, MRL fibroblast-like cells have a distinct cell-cycle (G2/M accumulation) phenotype and a heightened basal and wound site DNA damage/repair response that is also common to classical regenerators and mammalian embryonic stem cells. Additionally, a neutral and alkaline comet assay displayed a persistent level of intrinsic DNA damage in cells derived from the MRL mouse. Similar to mouse ES cells, the p53-target p21 was not expressed in MRL ear fibroblasts. Because the p53/p21 axis plays a central role in the DNA damage response and cell cycle control, we directly tested the hypothesis that p21 down-regulation could functionally induce a regenerative response in an appendage of an otherwise nonregenerating mouse strain. Using the ear hole closure phenotype, a genetically mapped and reliable quantitative indicator of regeneration in the MRL mouse, we show that the unrelated Cdkn1a tmi/Tyj /J p21 −/− mouse (unlike the B6129SF2/J WT control) closes ear holes similar to MRL mice, providing a firm link between cell cycle checkpoint control and tissue regeneration.

MRL mice display unusual healing properties. When MRL ear pinnae are hole punched, the holes close completely without scarring, with regrowth of cartilage and reappearance of both hair follicles and sebaceous glands. Studies using (MRL/lpr x C57BL/6)F(2) and backcross mice first showed that this phenomenon was genetically determined and that multiple loci contributed to this quantitative trait. The lpr mutation itself, however, was not one of them. In the present study we examined the genetic basis of healing in the Large (LG/J) mouse strain, a parent of the MRL mouse and a strain that shows the same healing phenotype. LG/J mice were crossed with Small (SM/J) mice and the F(2) population was scored for healing and their genotypes determined at more than 200 polymorphic markers. As we previously observed for MRL and (MRL x B6)F(2) mice, the wound-healing phenotype was sexually dimorphic, with female mice healing more quickly and more completely than male mice. We found quantitative trait loci (QTLs) on chromosomes (Chrs) 9, 10, 11, and 15. The heal QTLs on Chrs 11 and 15 were linked to differential healing primarily in male animals, whereas QTLs on Chrs 9 and 10 were not sexually dimorphic. A comparison of loci identified in previous crosses with those in the present report using LG/J x SM/J showed that loci on Chrs 9, 11, and 15 colocalized with those seen in previous MRL crosses, whereas the locus on Chr 10 was not seen before and is contributed by SM/J.

The MRL/MpJ mouse is an inbred laboratory strain of Mus musculus, known to exhibit enhanced autoimmunity, increased wound healing, and increased regeneration properties. We report the full-length mitochondrial DNA (mtDNA) sequence of the MRL mouse (Accession # EU450583), and characterize the discovery of two naturally occurring heteroplasmic sites. The first is a T3900C substitution in the TPsiC loop of the tRNA methionine gene (tRNA-Met; mt-Tm). The second is a heteroplasmic insertion of 1-6 adenine nucleotides in the A-tract of the tRNA arginine gene (tRNA-Arg; mt-Tr) at positions 9821-9826. The level of heteroplasmy varied independently at these two sites in MRL individuals. The length of the tRNA-Arg A-tract increased with age, but heteroplasmy at the tRNA-Met site did not change with age. The finding of naturally occurring mtDNA heteroplasmy in an inbred strain of mouse makes the MRL mouse a powerful new experimental model for studies designed to explore therapeutic measures to alter the cellular burden of heteroplasmy.

Whereas amphibians regenerate lost appendages spontaneously, mammals generally form scars over the injury site through the process of wound repair. The MRL mouse strain is an exception among mammals because it shows a spontaneous regenerative healing trait and so can be used to investigate proregenerative interventions in mammals. We report that hypoxia-inducible factor 1α (HIF-1α) is a central molecule in the process of regeneration in adult MRL mice. The degradation of HIF-1α protein, which occurs under normoxic conditions, is mediated by prolyl hydroxylases (PHDs). We used the drug 1,4-dihydrophenonthrolin-4-one-3-carboxylic acid (1,4-DPCA), a PHD inhibitor, to stabilize constitutive expression of HIF-1α protein. A locally injectable hydrogel containing 1,4-DPCA was designed to achieve controlled delivery of the drug over 4 to 10 days. Subcutaneous injection of the 1,4-DPCA/hydrogel into Swiss Webster mice that do not show a regenerative phenotype increased stable expression of HIF-1α protein over 5 days, providing a functional measure of drug release in vivo. Multiple peripheral subcutaneous injections of the 1,4-DPCA/hydrogel over a 10-day period led to regenerative wound healing in Swiss Webster mice after ear hole punch injury. Increased expression of the HIF-1α protein may provide a starting point for future studies on regeneration in mammals.

The apolipoprotein E (ApoE) gene has been linked to maladies such as hypercholesterolemia, CNS injury and disease. In this study, we present evidence that, in addition to the known transcript (ApoE S1) that translates into ApoE, there are three additional transcripts in mice. Two of these transcripts, ApoE S2 and ApoE S3, which are predicted to be transmembrane proteins, are transcribed from the sense strand. ApoE AS1 is transcribed from the antisense strand and is complementary to exon 4 of ApoE S1. The open reading frame of ApoE AS1 is conserved between human and mouse. The antisense transcript falls within the region of the human epsilon 4 allele that has been linked to the familial onset form of Alzheimer's disease. We also demonstrate the expression of ApoE S3 and ApoE AS1 in ApoE knockout mice, and ApoE S1 and ApoE S2 do not get transcribed. We had previously identified ApoE S1 as being upregulated in mice after spinal cord injury. In this study, we show that in spinal cord-injured C57BL/6 mice, both ApoE S1 and ApoE S3 transcripts are 10-fold upregulated and the antisense ApoE AS1 is 100-fold upregulated compared with normal levels. Such data suggest that these alternate transcripts are involved in the molecular pathogenesis of CNS disease and perhaps in ApoE expression in general, as we show that ApoE S2 and AS1 are also transcribed in human.

We previously demonstrated that after a severe cryoinjury to the right ventricle of the heart, adult MRL mice display structural and functional recovery with myocardial tissue replacement resembling that seen in amphibians. The control non-regenerating adult C57BL/6 (B6) mouse shows a predominant scar response. In the present study, radiation chimeras reconstituted with fetal liver cells from either healer MRL or nonhealer B6 mice were generated to test for a transfer of phenotype. Allogeneic MRL fetal liver cells were injected into x-irradiated (9 Gy) B6 mice and B6 fetal liver cells were injected into x-irradiated MRL mice. In these allogeneic chimeras, the healing response to cardiac cryoinjury was predominantly of the donor phenotype. Thus, MRL fetal liver cells transferred the healing phenotype to the B6 nonhealer with the appearance of Y-chromosome positive, donor-derived cardiomyocytes in the injury site and MRL-like healing with little scar. Similarly, B6 fetal liver cells transferred the nonhealing phenotype to the MRL with little cardiomyocyte growth and an acellular B6-like scar. These results are in contrast to the ear hole closure response which was of the recipient phenotype. We conclude that, in the case of the heart, fetal liver-derived stem cells regulate regenerative healing.

The MRL mouse has been shown to display an epimorphic regenerative response after ear hole punching leading to complete closure within 30 days and cartilage regrowth. The regenerative capacity of the MRL has also been seen after a severe cryoinjury to the heart leads to complete healing without scarring and functional myocardium. The wound healing ear hole closure response that occurs in MRL mice has been shown to be genetically controlled. We have previously identified 11 quantitative trait loci (QTL) that govern healing in an intercross of (MRL x C57BL/6 J) mice. However, it is desirable to use another poorly healing mouse strain to elucidate the full range of genetic factors that affect this important process. In the current study, we have used an inbred subspecies of the mouse, M. castaneus, and have confirmed a number of loci identified previously. In addition, we report three new healing QTL. Furthermore, in this strain combination, we note a strong sexual dimorphism also observed in the MRL x C57BL/6 cross, both in the healing trait and in the QTL that control it.

The ability to regenerate tissues and limbs in its most robust form is seen in many non–mammalian species. The serendipitous discovery that the MRL mouse has a profound capacity for regeneration in some ways rivalling the classic newt and axolotl species raises the possibility that humans, too, may have an innate regenerative ability. The adult MRL mouse regrows cartilage, skin, hair follicles and myocardium with near perfect fidelity and without scarring. This is seen in the ability to close through–and–through ear holes, which are generally used for lifelong identification of mice, and the anatomic and functional recovery of myocardium after a severe cryo–injury. We present histological, biochemical and genetic data indicating that the enhanced breakdown of scar–like tissue may be an underlying factor in the MRL regenerative response. Studies as to the source of the cells in the regenerating MRL tissue are discussed. Such studies appear to support multiple mechanisms for cell replacement.

Preadipocyte factor-1 (Pref-1), a delta-like protein containing epidermal growth factor-repeats, is expressed in proliferating cells in a variety of tissues and is believed to be involved in maintaining the undifferentiated state of these cells. Using microarray analysis, reverse transcriptase-polymerase chain reaction, in-situ hybridization, and immunohistochemistry, we have identified Pref-1 expression in the healing ears of two strains of mice, MRL and C57BL/6. MRL is unusual in that ear punches completely regenerate the ear tissue along with new cartilage with no scarring. Pref-1 is more highly expressed in the MRL wounds, is uniquely found in a condensation of cells within the regenerating tissue of the blastema, and may contribute to the regenerative capacity of the MRL ear wound.

Objective: The wound healing response may be viewed as partially overlapping sets of two physiological processes, regeneration and wound repair with the former overrepresented in some lower species such as newts and the latter more typical of mammals. A robust and quantitative model of regenerative healing has been described in Murphy Roths Large (MRL) mice in which through-and-through ear hole wounds in the ear pinna leads to scarless healing and replacement of all tissue through blastema formation and including cartilage. Since these mice are naturally autoimmune and display many aspects of an enhanced inflammatory response, we chose to examine the inflammatory status during regenerative ear hole closure and observed that inflammation has a clear positive effect on regenerative healing. Approach: The inflammatory gene expression patterns (Illumina microarrays) of early healing ear tissue from regenerative MRL and nonregenerative C57BL/6 (B6) strains are presented along with a survey of innate inflammatory cells found in this tissue type pre and postinjury. The role of inflammation on healing is tested using a COX-2 inhibitor. Innovation and Conclusion: We conclude that (1) enhanced inflammation is consistent with, and probably necessary, for a full regenerative response and (2) the inflammatory gene expression and cell distribution patterns suggest a novel mast cell population with markers found in both immature and mature mast cells that may be a key component of regeneration.

The Golli-MBP transcription unit contains three Golli-specific exons as well as the seven exons of the classical myelin basic protein (MBP) gene and encodes alternatively spliced proteins that share amino acid sequence with MBP. Unlike MBP, which is a late Ag expressed only in the nervous system, Golli exon-containing gene products are expressed both pre- and postnatally at many sites, including lymphoid tissue, as well as in the central nervous system. To investigate whether Golli-MBP peptides unique to Golli would result in neurological disease, we immunized rats and observed a novel neurological disease characterized by mild paralysis and the presence of groups of lymphocytes in the subarachnoid space but not in the parenchyma of the brain. Disease was induced by Th1-type T cells that displayed an unusual activation phenotype. Primary stimulation in vitro induced T cell proliferation with increased surface CD45RC that did not become down-regulated as it did in other Ag-stimulated cultures. Secondary stimulation of this CD45RChigh population with Ag, however, did not induce proliferation or IL-2 production, although an IFN-γ-producing population resulted. Proliferation could be induced by secondary stimulation with IL-2 or PMA-ionomycin, suggesting an anergic T cell population. Cells could adoptively transfer disease after secondary stimulation with IL-2, but not with Ag alone. These responses are suggestive of a chronically stimulated, anergic population that can be transiently activated to cause disease, fall back into an anergic state, and reactivated to cause disease again. Such a scenario may be important in chronic human disease.

Wound healing of mammalian tissue is an essential process in the maintenance of body integrity. The general mechanism of wound healing usually studied in adult mammals is repair, in contrast to the regeneration seen in more primitive vertebrates. We recently have discovered that MRL/MpJ mice, unlike all other strains of mice tested, undergo rapid and complete wound closure that resembles regeneration. Specifically, through-and-through surgical ear hole wounds close without scarring in Fas lpr × C57BL/6)F2 and backcross populations. In the primary screens of these populations, quantitative trait loci that control the extent of wound closure were detected on chromosomes 8, 12, and 15 and at two separate locations on chromosome 13. Evidence of further genetic control of healing was found on chromosome 7. All alleles that contribute to full wound closure are derived from the MRL/MpJ- Fas lpr parent except for the quantitative trait locus on chromosome 8, which is derived from C57BL/6.

Neuritogenic T cells specific for SP-26, a synthetic peptide (residue 53-78) of myelin P2 protein that causes experimental autoimmune neuritis (EAN), use the same T cell receptor (TCR) V gene family (V beta 8) that can induce experimental autoimmune encephalomyelitis (EAE) in Lewis rats. Tolerance to autoregulatory T cells may be induced in rats by intravenous (iv) administration of antigen-coupled splenocytes; however, the mechanisms that lead to altered immune reactivity are not well understood. Here we demonstrate that SP-26, when coupled to syngeneic spleen cells and administered iv, either before or after disease induction, markedly inhibited development and expression of clinical signs and histological changes of EAN. The induction of tolerance by this method was peptide-specific and MHC-restricted. We showed previously that T cells involved in EAN utilize the T cell antigen receptor V beta 8, whereas less than 5% of normal rat peripheral T cells express V beta 8. We have examined T lymphocytes from tolerized rats to determine the presence or absence of V beta 8(+)-bearing cells in order to determine the mechanism of tolerance. V beta 8 cells were undetectable by Northern blot analysis in the lymph nodes of unimmunized animals but easily detected in SP-26-primed and tolerized rats. In addition, spleen cells isolated from tolerized animals were anergic and failed to proliferate in response to SP-26, but retained responsiveness to IL-2 and Con A stimulation. Thus, the peptide-specific unresponsiveness that can be induced in rats with EAN, a T-cell-mediated process that is MHC-restricted and utilizes the T cell receptor V beta 8, occurs while V beta 8 transcripts remain readily detectable in spleen and lymph node cells. The detection of V beta 8-bearing T cells requires the development of antibodies specific for this rat surface protein.

Experimental allergic neuritis, an autoimmune disease of the peripheral nervous system, is a model for human Guillain-Barré syndrome. Experimental allergic neuritis is mediated by CD4+ T cells reactive with myelin P2 protein. We demonstrate that these T cells use the same members of T-cell receptor V gene families for both their alpha (V alpha 2) and beta (V beta 8) chains as T cells that cause experimental allergic encephalomyelitis, an autoimmune disease of the central nervous system. Furthermore, these T cells appear to be idiotypically related. Therefore, completely different T-cell lines with different antigen specificities, producing entirely different diseases, share common T-cell receptors.

Digit re-growth following amputation injury proximal to the first phalangeal joint is not a property of mammalian wound healing. However, the regenerative potential observed in the MRL mouse invites a re-examination of this rule. In this study, healing was assessed in three mouse strains after amputation midway through the second phalangeal bone. Three distinct outcomes were observed though evidence for re-growth was observed only in the MRL mouse. Here, a blastema-like structure was seen along with apparent chondrogenesis, consistent with a histological profile of a regenerative response to injury. Analysis of trichrome staining and basement membrane changes, proliferation and apoptosis indicated that these processes contributed to the formation of new digit tissue. On the other hand, SW and B6 digits did not show evidence of growth with little mesenchymal BrdU incorporation or phosphorylation of H3.

A particular mouse strain, the MRL mouse, has been shown to have unique healing properties that show normal replacement of tissue without scarring. The serendipitous discovery that the MRL mouse has a profound capacity for regeneration in some ways rivaling the classic newt and axolotl species raises the possibility that humans, too, may have an innate regenerative ability. We propose this mouse as a model for continuous regeneration with possible life-extending properties. We will use the classical "immortal" organism, the hydra, for comparison and examine those key phenotypes that contribute to their immortality as they are expressed in the MRL mouse versus control mouse strains. The phenotypes to be examined include the rate of proliferation and the rate of cell death, which leads to a continual turnover in cells without an increase in mass.

The MRL mouse was proposed as a model of mammalian regeneration because it can close ear holes completely with the restoration of normal tissue. This regeneration process involves the formation of a blastema during healing, the re-appearance of cartilage and hair follicles, and healing without scarring. Such a process requires extensive tissue remodeling. To characterize differences in ear wounding responses between regenerating and nonregenerating mice, we examined and compared the extracellular matrix remodeling and the matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinase (TIMP) response in the MRL and C57BL/6 mouse strains after injury. We found a correlation between the MRL's ability to break down the basement membrane, form a blastema, and close ear hole wounds and an inflammatory response with neutrophils and macrophages seen in the ear after injury. These cells were positive for MMP-2 and MMP-9 as well as TIMP-2 and TIMP-3. Clear differences between the MRL and B6 response to injury were seen that could explain the differences in healing and blastema formation in the MRL and lack of it in the B6 mice. This finding was further supported by enzyme activity as determined by gelatin zymography.

The MRL mouse has been shown to display unusual healing properties. In particular, when the ear pinna is hole punched, the hole that is made closes completely without scarring, with reformation of hair follicles and sebaceous glands, and regrowth of cartilage. Initial studies using (MRL/lpr × C57BL/6) F2 and backcross mice showed that this phenomenon is genetically determined and that multiple loci contribute to this quantitative trait. In the present study, with twice as many animals, we have confirmed many of the original heal loci and identified new ones. We have also found that this phenotype is sexually dimorphic in that female mice heal more quickly and more completely than male mice. To test the cause of this difference, we castrated both males and females. Castration of males led to better healing, although ovariectomy did not lead to worse healing in female mice. Finally, most heal loci were shown to be responsible for regulating healing primarily in male animals more than in females, or vice versa. Thus, sex plays a highly significant role in the closure of wounded tissue in this mammalian model of healing and regeneration.

Regeneration is generally considered to be a phenomenon restricted to amphibians in which amputated limbs reform and regrow. We have recently noted a strain of mouse, the MRL, which displays a remarkable capacity for cartilagenous wound closure and provides an example of a phenomenon previously considered to be a form of regeneration. Specifically, through-and-through ear punches rapidly attain full closure with normal tissue architecture reminiscent of regeneration seen in amphibians as opposed to scarring, as usually seen in mammals. Histologically, we have demonstrated normal cell growth and microanatomy, including angiogenesis and chondrogenesis, as opposed to control C57BL/6 mice which have ear holes that contract minimally but do not close. Finally, this phenomenon is a genetically definable quantitative trait.