As ear hole closure has been best studied in young MRL mice, we wanted to know if events occurring in our older B6 mice resembled what had previously been described in young “autoimmune” MRL mice.6
We first examined the overall macroscopic appearance of closed holes in young MRL and older B6 mice at 1 month postpunch to look for any obvious differences. None were apparent. In both strains, holes filled so completely with new tissue that it was sometimes difficult to find the site of the original hole ().
FIG. 1. Comparison of macroscopic appearance of complete closure of 2.2-mm holes in MRL and B6 mice. Ears of MRL mice (albino) were punched with a 2.2-mm cutter at 2 months of age, whereas B6 ears (black) were punched at 9 months of age. The appearance of the (more ...)
To evaluate more fully the effect of age on the regenerative capacity of conventional (non-“autoimmune”) strains of mice, we compared 2-mm ear-hole closure rates in B6 and BALB/c mice that were punched at different ages. At all ages, the rate of closure seems to have two phases, an early rapid phase that lasts for about 2 weeks, followed by a much slower phase, during which the process is gradually completed (). Although these two phases are characteristic of all ages, the details differ with age. The initial (“rapid”) phase was quite slow in mice punched at 1 month and increased with age. In addition, young mice never completed the process of macroscopic hole closure (BALB/c mice averaged 40%, and B6 averaged 65% replacement tissue), whereas mice punched at 8–9 months averaged 90–97%; many of them closed completely.
, shows the percent hole closure at 14 days postpunch for mice that were punched at different ages. We chose day 14 because most growth had occurred by this point. For both BALB/c and B6 mice, ear closure improved with age, peaking at 8–10 months and then declining somewhat at 12 months. We conducted ANOVA on the 1–8 months at punch data for both strains and found statistically significant differences between the 1–2 months and middle-aged months at punch (see legend to for details).
To determine if the biological processes underlying the ear closure in middle-aged B6 and BALB/c mice are similar to the well-known regeneration of young MRL mice, or if the older non-autoimmune mice were simply filling the holes with fibrotic scar tissue, we undertook histological analysis. compares the appearance of ear holes at 120 days postpunch from an MRL and a B6 mouse punched at 2 and 8 months of age, respectively. The holes have filled in similarly, with regrowth of adipocytes, chondrocytes, sebaceous glands (), and hair follicles. The main difference in the appearance of regenerated ear tissue in MRL and B6 mice is that melanocytes have also regenerated in the dermis of the B6 mouse. That these elongated brown cells, which appear in all B6 histopathology images at all ages at punch, were not seen in MRL and are indeed melanocytes was confirmed by inspection at 400× magnification (Supplemental Figs. 2 and 3
). Thus, the hole closure in middle-aged B6 mice is indeed regenerative.
FIG. 3. Similar histological appearance of regenerated ear hole tissues in middle-aged B6 and young post-weaning MRL mice at 4 months postpunch. Ears of B6 mice that had been punched at 8 months of age, and MRL mice punched at 2 months of age were examined at (more ...)
To examine what histological differences might underlie the age-related improvement in regeneration, we examined the regenerating tissues of increasingly old B6 mice at day 6 after punch, using both H & E bright-field illumination (where protein appears pink) and fluorescence imaging (where the eosin/protein complex glows yellow). The most obvious age-related difference in B6 was in the thickness of the early apical epidermis (), which at 9 months at punch was much like the classical “apical epidermal cap” seen in MRL mice () and in amphibian limb regeneration. Young B6 mice developed thinner apical caps. In other respects, both young and old B6 mice showed strikingly amphibian-like regenerative features. Under bright-field illumination, beginning at about day 3 postpunch, a bluish-staining primitive “myxomatous” connective tissue appeared under the new epithelium, instead of the pink-staining granulation tissue that precedes scar formation. Using fluorescence imaging, the myxomatous tissue appeared as a black region with no basement membrane above it, suggesting that these regions were low in protein and high in carbohydrate (most likely hyaluronate13
), similar to the myxomatous matrix of amphibians and embryos. At all ages, the regenerating epidermis generated downgrowths oriented toward the mature cartilage (demarcated by a white circle), a feature similar to classical embryonic epithelial–mesenchymal interactions. Thus, regeneration is not limited to one “autoimmune” mouse strain, but occurs in common non-autoimmune strains, improves with age, and exhibits the stages of classical amphibian epimorphic regeneration (Supplemental Figs. 4 and 5
That MRL mice are not alone in their ability to regenerate had been hinted at earlier. Geneticists looking for the genes that might govern the MRL mouse's ability to regenerate studied the four founder strains from which the MRL mouse was created, and found that, of the four, the “Large” (LG) mouse also had the ability to completely close experimental ear holes.15
Because the MRL mouse is also an extremely large mouse, we were intrigued by the possibility that size might correlate with early regeneration ability. In the only previous study to explore the influence of body size on ear hole closure, Li et al. compared the rate
of ear-hole closure of several strains punched at 5 weeks and concluded that body weight had little influence.15
We revisited this question, comparing instead, the extent
of closure at a single late time point (day 30) with adult mouse strain body weight at 6 months. We obtained our closure data directly from Li, as a colored graph of from their paper. For body weight data, we used the average adult weight (including males and females) of each strain.16–18
shows the comparison for 18 of the 26 strains in the Li study for which body weight data at 6 months were available, revealing that there is indeed a correlation between average adult body weight and the extent of hole closure.
FIG. 5. Correlation of adult body weight and hole closure. Using hole closure data from Li et al.14 and body weight data from the Jackson Laboratories Phenome database,16,17 we examined the correlation between hole closure and body weight, plotting the average (more ...)