ABSTRACT
Amphibian forelimb regeneration is a nervedependent process; nerves presumably release one or more neurotrophic factors that stimulate blastema cell division. To date several candidate molecules/factors have been shown to stimulate macromolecular synthesis and/or mitosis but sustained cell cycle activity and blastema development have not been achieved. Because dorsal root ganglia (DRG) implants are capable of promoting regeneration of denervated adult newt limbs (Kamrin & Singer, 1959), we have evaluated the DRG stimulation of regeneration in denervated limbs of adult newts and larval axolotls; two alternative timing strategies were tested as a step toward defining bioassay parameters that best reflect neurotrophic activity. The frequency of regeneration in denervated adult newt limbs was compared after providing DRG before or at the time of denervation (to maintain neurotrophic and cell cycle activity) versus DRG implantation at various postdenervation times (to resupply neurotrophic activity and restimulate suppressed cell cycle activity).
The results show that denervated adult newt limbs regenerated most frequently using the maintenance strategy, but as the denervation interval was extended in the restimulation strategy, the frequency of regeneration declined. Larval axolotl limbs responded positively in both maintenance and restimulation DRG-grafting protocols. These results suggest that the efficacy of DRG stimulation of regeneration in adult newts was related to the relative number of blastema cells present at the time of denervation and the proliferative status of the blastema cells; bioassays with denervated adult newt limbs should be designed with these constraints in mind. Because such constraints are not as problematic with the larval axolotl, this species may provide the best opportunity for further defining bioassay parameters related to the neurotrophic stimulation of regeneration.
Introduction
Regeneration of the urodele forelimb is dependent upon an adequate nerve supply (reviewed by Singer, 1952; Wallace, 1981). While the precise biochemical nature of the neurotrophic influence and mechanism of nerve action are as yet not defined, progress has been reported towards definitive resolution of these problems. Subsequent to denervation, macromolecular synthesis (Dresden, 1969; Lebowitz & Singer, 1970; Singer & Caston, 1972) and mitosis (Singer & Craven, 1948; Mescher & Tassava, 1975; Globus, 1978; Tomlinson, Globus & Vethamany-Globus, 1984) are suppressed and it has been speculated that the neurotrophic influence promotes sustained cell cycling by stimulating events in either the G1 (Globus, 1978; Maden, 1978) and/or G2 (Tassava & Mescher, 1975) phases of the cell cycle. Appropriately therefore, DNA and protein synthesis as well as mitotic and [3H]thymidine labelling indices have been used as bioassay parameters to evaluate the efficacy of treating denervated limbs with putative neurotrophic factors. In fact, increases in blastema macromolecular synthesis, or mitotic and DNA labelling indices have been reported following treatment with dorsal root ganglia (Globus & Vethamany-Globus, 1977; Vethamany-Globus, Globus & Tomlinson, 1978; Vethamany-Globus, Globus, Darch, Milton & Tomlinson, 1984; Tomlinson, Globus & Vethamany-Globus, 1981), nerve extracts (Lebowitz & Singer, 1970; Singer, Maier & McNutt, 1976; Jabaily & Singer, 1977; Carlone & Foret, 1979; Mescher & Loh, 1981) and purified molecules (Mescher & Gospod-arowicz, 1979; Globus, Vethamany-Globus, Kesik & Milton, 1983; Mescher & Muniam, 1984; Carlone & Rathbone, 1985).
The ultimate demonstration of neurotrophic activity will be the promotion of blastema development on a denervated limb (Wallace, 1981; Olsen, Barger & Tassava, 1984). Since blastema growth clearly requires the coordinated syntheses of DNA, RNA and proteins which culminate in mitotic divisions, it is understandable that intermittent treatment of denervated limbs with purified molecules and/or extracts has at present resulted in only partial recovery of these parameters; no single chemical treatment has resulted in growth of a regenerate (reviewed by Wallace, 1981). In this regard it is noteworthy that dorsal root ganglia (DRG), isolated from their central nervous system connections and grafted into denervated limbs, are capable of stimulating blastema growth and regeneration (Kamrin & Singer, 1959). As a potentially complete source of neurotrophic factor(s) ganglia therefore provide the opportunity to examine the relevance of the various bioassay parameters currently employed. In the Kamrin and Singer study, DRG were excised between 10 and 13 days postamputation (thereby denervating the limb) and simultaneously autografted to the distal limb tissues. These early regenerates were technically never denervated since the implanted DRG presumably maintained uninterrupted neurotrophic activity. More recent studies using nerve extracts or isolated molecules have not followed the timing protocol used by Kamrin and Singer. Instead, treatment was administered 48 h or more after denervation, at times when macromolecular synthesis (Singer & Caston, 1972) and mitosis (Singer & Craven, 1948) were already reduced. The reported small increases in bioassay parameters (reviewed by Wallace, 1981) therefore represent only minimal stimulation by neurotrophic activity. Perhaps the introduction of an interval between denervation and treatment with a putative neurotrophic factor is responsible for the minimal stimulations that have been observed.
In the present study we used the DRG-implantation technique (Kamrin & Singer, 1959) to investigate two alternative timing strategies with respect to the neurotrophic stimulation of regeneration in denervated limbs. We tested the hypothesis that as the denervation interval increases, the regenerates become committed to an alternative fate (i.e. differentiation or resorption) and no longer respond to neurotrophic stimulation. We compared the frequency of regeneration in denervated limbs after providing DRG implants before, or at the time of, denervation (maintenance of neurotrophic and cell cycle activity) versus implantation at various post denervation times (resupplying neurotrophic activity to restimulate suppressed cell cycle activity). The results showed that denervated adult newt limbs regenerated most successfully using the maintenance strategy and that as the length of the denervation interval was extended to 1 and 2 days, successful DRG stimulation of regeneration progressively declined.
Neither adult newt nor larval axolotl limbs regenerate after denervation; however, axolotl limbs are unique in that following regrowth of the transected nerves, limbs spontaneously regenerate without reamputation (Schotté & Butler, 1941; Petrosky, Tassava & Olsen, 1980). Ingrowing nerve fibres stimulate cells previously arrested in the cell cycle (Olsen et al. 1984) but more detailed cell cycle studies are impractical since the timing of reinnervation exhibits considerable variability (10-14 days after denervation; Olsen et al. 1984). DRG grafts, previously untested in denervated larval axolotl limbs, provided a means of specifically timing neurotrophic stimulation. We therefore examined the response of denervated larval axolotl limbs to xenografts of adult newt DRG, again using maintenance and restimulation strategies. DRG were grafted either concomitant with denervation (maintenance) or following various denervation intervals (restimulation). Unlike adult newt limbs, denervated larval axolotl limbs responded positively to both maintenance and restimulation DRG-grafting protocols.
Materials and methods
Larval axolotls (Ambystoma mexicanum), of 50–60 mm body length, and adult newts (Notophthalmus viridescens) were anaesthetized in neutralized 0 · 15 % MS 222 and bilaterally amputated through either the proximal third of the radius and ulna (larval axolotls) or distal humerus (adult newts). Due to differences in the size of forelimbs and hindlimbs, only forelimbs were used, thereby providing a built-in control for size within each experimental series. Since nerve regeneration is very rapid in the larval axolotl (10 – 14 days, Olsen et al. 1984) compared to adult newts (14 days, Salley & Tassava, 1981), more distal amputation planes were used in axolotls in order to increase the time period over which the axolotl limbs remained denervated. Limbs were denervated by transection of the third, fourth and fifth brachial nerves and redenervated at 8-day intervals in axolotls and at 2-week intervals in newts. The completeness of denervation was confirmed for every case by the total absence of motor and sensory function, by the failure of all denervated limb stumps to form a regeneration blastema and by the presence of only degenerated nerve fragments in histological sections of denervated limbs stained for nerves according to the method of Samuel (1953).
Implant procedure
Dorsal root ganglia (the paired 3rd and 4th brachial and the 16th and 17th crural), pituitary glands, flank muscle or liver, obtained from decapitated newts, were temporarily (15 min maximum) stored in chilled Holtfreter’s solution. A small ventral incision was made in the limb skin at a level just proximal to the original plane of amputation and, using a single tyne of a pair of watchmaker’s forceps, a tunnel was created through the underlying tissues to the distal end of the limb. Either control tissue or randomly selected brachial or crural ganglia were then inserted into the tunnel and positioned under the margin of the skin and wound epithelium. The DRG were obtained solely from newts so that a single source of neurotrophic factors could be evaluated.
Adult newt control limbs received pituitary gland homografts and larval axolotl control limbs were implanted with ganglion-sized newt flank muscle or liver. Additional control limbs were either denervated and sham implanted or remained normally innervated. The different implantation timing protocols used in each experiment are illustrated in Fig. 1.
Timing protocol of experiments designed to test the maintenance and restimulation strategies for the promotion of regeneration on denervated urodele limbs by grafted dorsal root ganglia (DRG). Adult newt forelimbs were denervated (represented by the dotted vertical line) 1 (Series I), 10 (Series II) or 14 days (Series HI) after amputation. Larval axolotl limbs were denervated 1 (Series IV) or 4 (Series V) days after amputation. In the maintenance strategy, DRG were grafted either 2 days before or at the time of denervation (represented by the number on, or to the left of, the vertical line) and in the restimulation strategy, DRG were grafted on various days after denervation (represented by numbers to the right of the vertical line).
Timing protocol of experiments designed to test the maintenance and restimulation strategies for the promotion of regeneration on denervated urodele limbs by grafted dorsal root ganglia (DRG). Adult newt forelimbs were denervated (represented by the dotted vertical line) 1 (Series I), 10 (Series II) or 14 days (Series HI) after amputation. Larval axolotl limbs were denervated 1 (Series IV) or 4 (Series V) days after amputation. In the maintenance strategy, DRG were grafted either 2 days before or at the time of denervation (represented by the number on, or to the left of, the vertical line) and in the restimulation strategy, DRG were grafted on various days after denervation (represented by numbers to the right of the vertical line).
Differences in the timing of denervation and DRG implantation were required in order to compensate for different rates of limb and nerve regeneration between these species. The time frame of each experimental series was adjusted to compensate for the aforementioned faster rate of nerve regeneration and the fact that innervated limbs of larval axolotls can produce a blastema in 6 – 8 days postamputation whereas adult newts require 14 – 18 days to form a similar stage regenerate (Tomlinson, Goldhamer, Barger & Tassava, 1985). If limbs are denervated at the time of amputation (both in newts and larval axolotls) limited cell division occurs for only a brief period of time (Mescher & Tassava, 1975; Maden, 1978; Olsen et al. 1984). Limbs denervated during the process of regeneration generally experience one of two fates (Singer & Craven, 1948; Butler & Schotté, 1949); with relatively early denervations the blastema resorbs and the limb fails to regenerate, but when denervation is delayed to the midbud stage or later, small hypomorphic spikes or small but complete limbs may result (a phenomenon known as nerve-independence). Denervation of blastema-stage regenerates results in a gradual reduction of cell division over a 2-day interval (Tassava & McCullough, 1978; Tomlinson et al. 1984).
Timing of DRG homografts into newt forelimbs
To examine the response of different-sized blastema cell populations to the stimulation by homografted DRG, limbs were implanted concomitant with denervation, either 1 (Series I), 10 (Series II) or 14 (Series III) days after amputation (Fig. 1).
To evaluate maintenance versus restimulation strategies, a pair of DRG were implanted at different times with respect to denervation in Series II and III (Fig. 1). In the maintenance strategy DRG were implanted either 48 h before or concomitant with denervation whereas in the restimulation strategy, DRG were grafted 24, 48 and in some cases 96 h after denervation.
Timing of DRG xenografts into axolotl forelimbs
Series IV
To maintain uninterrupted neurotrophic activity, a single DRG was grafted at the time of denervation (24 h after amputation). In denervated axolotl limbs dedifferentiated cells participate in limited cell division 4 to 6 days after amputation, whereas between 7 and 10 days postdenervation, proliferative activity is reduced to basal levels (Maden, 1978; Olsen et al. 1984). DRG were implanted 4 days after denervation (day 5 postamputation) in an attempt to maintain the aforementioned limited cell cycle activity. To restimulate cell division, DRG were implanted 8 days after denervation (9 days postamputation).
Series V
Axolotl limbs were denervated 4 days after amputation and a pair of DRG were xenografted concomitant with (maintenance strategy), or 4 days after denervation (restimulation strategy).
Staging and histology
Progressive changes were monitored by staging the regenerates (Iten & Bryant, 1973) at 2-day intervals. To compare with Kamrin & Singer (1959), limbs that regenerated to at least the midcone stage within the time constraints of the experiment (35 days postamputation for newts and 16 days postamputation for axolotl limbs) were tabulated as successful regenerates. The frequency of regeneration between implant groups was compared using a Chi-squared test for the equality of binomial proportions.
At the end of the experimental period, four or more limbs per group were fixed in Bouin’s fluid, embedded in paraffin and longitudinally sectioned at 10 μm. One set of representative sections from each limb was stained with Delafield’s haematoxylin and counterstained with eosin, and to confirm denervation a second group of representative sections was stained for nerves (Mescher & Tassava, 1975; Olsen & Tassava, 1984) by the Samuel (1953) technique.
Results
Homografts to denervated newt limbs
Controls
10 days after amputation innervated limbs showed no visible outgrowth, but mound-shaped regenerates were present 14 days after amputation and by 35 days regenerates ranged from midcone to digit stages (Table 1); the range of stages observed is a consequence of normal variation (Iten & Bryant, 1973).
No regeneration was observed when control limbs were denervated 1 or 10 days after amputation. However, because one consequence of increasing the postamputation age of the regenerates prior to denervation is the accumulation of a larger blastema cell population, denervation 14 days postamputation permitted the subsequent development of several very small spike regenerates (Table 1). In Series III some regenerates had therefore become nerve-independent (Singer & Craven, 1948) at the time of denervation (14 days postamputation). Comparable tiny amorphic spikes, also observed on some of the ganglia implanted limbs of Series III, were not counted as DRG-stimulated regenerates.
Data in Table 1 and Fig. 2 also show that none of the denervated limbs regenerated in either of the pituitary-implanted control groups. The voracious appetite, smooth moist skin and darkened pigmentation indicated that the ectopically located pituitary glands continued to function (Tassava, 1969). Therefore, the stimulation of regeneration in denervated newt limbs (reported below) can be directly attributed to the influence of grafted DRG.
A histogram showing the percentage of adult newt limbs that regenerated in Series II (denervation 10 days postamputation; scored 35 days postamputation). The open histogram bar illustrates that all of the innervated (I) control limbs regenerated while the solid bars show that none of the denervated (D) and pituitary implanted control limbs regenerated. Cross-hatched bars represent the percentage of DRG-implanted limbs stimulated to regenerate. With increased intervals between denervation (day 10) and subsequent implantation (days 11, 12 and 14) the percentage of limbs stimulated to regenerate progressively declined. The number (n) of limbs in each group is given in parentheses.
A histogram showing the percentage of adult newt limbs that regenerated in Series II (denervation 10 days postamputation; scored 35 days postamputation). The open histogram bar illustrates that all of the innervated (I) control limbs regenerated while the solid bars show that none of the denervated (D) and pituitary implanted control limbs regenerated. Cross-hatched bars represent the percentage of DRG-implanted limbs stimulated to regenerate. With increased intervals between denervation (day 10) and subsequent implantation (days 11, 12 and 14) the percentage of limbs stimulated to regenerate progressively declined. The number (n) of limbs in each group is given in parentheses.
Maintenance strategy
Implanting DRG either prior to, or at the time of, denervation (1, 10 or 14 days postamputation) allowed the maintenance strategy to be examined in adult newt limbs that contained sequentially larger blastema cell populations at the time of nerve withdrawal. Limbs denervated and concomitantly implanted with DRG 1 day after amputation (Series I) did not regenerate, whereas in Series II, DRG grafted 48 h before or at the time of denervation (day 10), stimulated regeneration on 32 % arid 34 % of the limbs, respectively (Table 2, Fig. 2). In Series III the percentage of regenerating limbs increased further to 64 % and 57 % when DRG were homografted either 48 h before or at the time of denervation (day 14) respectively (Fig. 3). Therefore, with each increase in the interval between amputation and denervation a significantly greater percentage of limbs (P<0 · 05) was stimulated to regenerate using the maintenance strategy. Furthermore, in both Series II and III implanting DRG 48 h prior to denervation was as effective (P>0 · 05) as implanting DRG concomitant with denervation.
A histogram showing the percentage of adult newt limbs that regenerated in Series III (denervated 14 days postamputation). While all of the innervated limbs (I; open bar) regenerated, denervated controls (D; solid bar) did not. A significantly greater percentage of limbs (P<0 · 05) regenerated when ganglia were grafted (cross-hatched bars) using the maintenance (days 12 and 14) versus the restimulation (days 15 and 16) strategy. The number (n) of limbs in each group is given in parentheses.
A histogram showing the percentage of adult newt limbs that regenerated in Series III (denervated 14 days postamputation). While all of the innervated limbs (I; open bar) regenerated, denervated controls (D; solid bar) did not. A significantly greater percentage of limbs (P<0 · 05) regenerated when ganglia were grafted (cross-hatched bars) using the maintenance (days 12 and 14) versus the restimulation (days 15 and 16) strategy. The number (n) of limbs in each group is given in parentheses.
Restimulation strategy
To examine the effects of short denervation intervals on the ability of adult newt limbs to respond to neurotrophic restimulation, DRG were grafted 24,48 or 96 h after nerve withdrawal. Data in Figs 2 and 3 illustrate a trend of declining stimulation by DRG with increased periods of denervation. In Series II, the frequency of stimulation was 25%, 15% and 12% with 24, 48 and 96 h intervals between denervation (day 10) and DRG grafting, respectively. In Series III, when the ganglia were implanted 24 or 48 h after denervation (day 14), only 18 % and 14 % of the limbs regenerated, respectively. By increasing the interval between amputation and denervation (i.e. Series II versus III), there was no significant difference (P> 0 · 70) in the percentage of denervated limbs stimulated by DRG to regenerate using the restimulation strategy. Similarly, within each series, the percentage of limbs stimulated to regenerate declined as the interval between denervation and subsequent DRG implantation increased; however these declines were not statistically significant (P>0 · 40).
Comparison of the maintenance and restimulation strategies
When examined in terms of the appropriate timing of DRG implantation, the data in Figs 2 and 3 illustrate that denervated adult newt regenerates respond more frequently using the maintenance versus restimulation strategy. There was no significant difference (P = 0·10) in the response to maintenance and restimulation strategies in Series II. In Series III the use of the maintenance strategy resulted in a significantly greater percentage (P<0·001) of regenerating limbs compared to the results obtained using the restimulation protocol. The frequency of regenerate outgrowth was significantly greater compared to the denervated controls of each series (either with or without homografted pituitary glands) when DRG were implanted 48h before (Series II, P = 0 · 04; Series III, P = 0 001) or at the time of denervation (Series II, P = 0 · 03; Series III, P = 0 · 003). On the other hand, it is important to note that compared to denervated controls the frequency of regenerate outgrowth was not significantly stimulated when DRG were grafted 24h (Series II, P = 0 08; Series 111, P = 0 · 16), 48h (Series II, P = 0 · 20; Series III, P = 0 · 21), or 96 h (P = 0 · 25) after denervation.
The morphological stage attained also depended on the interval between denervation and DRG implantation. More advanced regenerates were observed when there was no interruption of neurotrophic stimulation and conversely, less-advanced regenerates were observed by prolonging the preimplantation denervation interval (Table 2). Series III data also show that longer delays between denervation and DRG grafting resulted in an increased frequency of very small spike regenerates in both denervated controls and DRG-implanted limbs. This suggests that DRG implants stimulated the growth of regenerates which, at the time of implantation, contained a relatively large blastema cell population bordering on nerve-independence. Moreover, the maintenance strategy was more effective in stimulating substantial regenerative outgrowth thus avoiding the minimal nerve-independent responses observed on denervated control limbs and some DRG-implanted limbs when the restimulation strategy was used.
Xenografts to denervated axolotl limbs
Controls
In Series IV and V, limbs that remained innervated throughout the experiment had blastema-stage regenerates 7 days postamputation and three-to four-digitstage regenerates 12 to 14 days postamputation. Of the limbs allowed to become reinnervated following a single denervation (n = 14), three progressed to the midcone stage within the 16-day experimental period; had these reinnervated limbs been fixed later, it is likely that a larger percentage would also have regenerated (Petrosky et al. 1980). Redenervated, nonimplanted and liver- or muscle-implanted control limbs did not regenerate (Table 3) but resorbed to the level of the proximal radius-ulna or distal humerus.
Maintenance and restimulation strategies
In Series IV, DRG xenografted at the time of denervation (24 h after amputation) to maintain neurotrophic stimulation, promoted blastema formation and regenerate outgrowth on 50 % of the implanted limbs (Table 4). DRG implanted 96 h after denervation, to maintain cell cycling and resupply neurotrophic activity, promoted regeneration on 90 % of the implanted limbs. When DRG were grafted 8 days after denervation, to restimulate cell cycling and resupply neurotrophic activity, 53 % of the limbs were stimulated to regenerate. At each time interval, implanted DRG promoted a significant stimulation of regeneration (P< 0 · 001) compared to denervated limbs (with or without control tissue implants). Limbs implanted with DRG on day 4 regenerated at a significantly greater frequency (P<0 · 04) than limbs implanted 8 days after nerve withdrawal. Comparisons between other implant groups did not show statistically significant differences (P>0 · 05).
The morphological stage attained varied with the timing of DRG implantation. Table 4 data show that the most advanced regenerates (palette and notch stages) were observed among limbs implanted 4 days after denervation. While two palette regenerates were observed on limbs implanted 8 days after denervation, midcone stage regenerates were most common and with concomitant denervation and DRG grafting only midcone stage regenerates were observed.
In Series V, regeneration occurred in five of six limbs implanted with a pair of DRG at the time of denervation (4 days after amputation). All five limbs reached at least the cone stage of regeneration and three of these progressed to the three-digit stage (Table 4); one limb did not retain the graft and resorbed. All six limbs that received DRG grafts 4 days after denervation regenerated to the midcone stage (Table 4).
Histology
All tissues expected in a mature limb (muscle, cartilage and connective tissue) were present in DRGstimulated regenerates; the DRG remained at or near the original plane of amputation (Figs 4, 5). In implanted adult newt limbs the homoplastically grafted DRG were invariably obscured by an accumulation of cells, probably the result of an immunological rejection of the graft; as a consequence neuronal cell numbers were substantially reduced (Fig. 4C,D).
Denervated adult newt forelimbs stimulated to regenerate following the implantation of two dorsal root ganglia (G). (A) A late-cone-stage regenerate formed following the implantation of ganglia 1 day after denervation (Series II). The bar represents 250μ. (B) This four-digit-stage regenerate developed following the implantation of ganglia at the time of denervation (Series HI). While two digits are present in this section, the remaining portions of the other digits are observed only in adjacent sections. The bar represents 250μm. (C) An enlargement of the box in (B) to show a portion of the region containing a representative implanted ganglion. Note the dense accumulation cf cells, which obscure the ganglia, and the reduced number of neuronal cell bodies (arrows). The bar represents 50gm. (D) A further enlargement of the ganglion in C to show the few neurones (arrows) found in the limb. The bar represents 20 μm. Compare the appearance of the ganglia implants in Fig. 4 to those in Fig. 5. In A and B the approximate plane of amputation (midhumerus) is marked by a dashed line. In C and D regeneration is directed toward the top of the micrograph. Haematoxylin and eosin staining.
Denervated adult newt forelimbs stimulated to regenerate following the implantation of two dorsal root ganglia (G). (A) A late-cone-stage regenerate formed following the implantation of ganglia 1 day after denervation (Series II). The bar represents 250μ. (B) This four-digit-stage regenerate developed following the implantation of ganglia at the time of denervation (Series HI). While two digits are present in this section, the remaining portions of the other digits are observed only in adjacent sections. The bar represents 250μm. (C) An enlargement of the box in (B) to show a portion of the region containing a representative implanted ganglion. Note the dense accumulation cf cells, which obscure the ganglia, and the reduced number of neuronal cell bodies (arrows). The bar represents 50gm. (D) A further enlargement of the ganglion in C to show the few neurones (arrows) found in the limb. The bar represents 20 μm. Compare the appearance of the ganglia implants in Fig. 4 to those in Fig. 5. In A and B the approximate plane of amputation (midhumerus) is marked by a dashed line. In C and D regeneration is directed toward the top of the micrograph. Haematoxylin and eosin staining.
Denervated axolotl forelimbs stimulated to regenerate following the implantation of a single dorsal root ganglion (G). (A) An early three-digit regenerate (digits are numbered 1, 2 and 3) formed following the implantation of a ganglion 1 day after denervation (Series IV). The approximate location of the ganglion (G; not seen in this section) is just distal to the plane of amputation (midradius-ulna; dashed line). The bar represents 200 μ m. (B) A ganglion (G) implanted 4 days after denervation (Series IV) stimulated the growth of this palette-stage regenerate. Note the large accumulation of blastema cells distal to the ganglion which is near the plane of amputation (dashed line). The bar represents 175 gm. (C) An enlargement of half of the ganglion in B showing the abundant neuronal cell bodies, some of which are indicated by arrows. The bar represents 100 μ m. (D) A further enlargement of the ganglion in C to show the abundant neurones (arrows) found in these limbs. The bar represents 5 μ m. In C and D regeneration is directed toward the top of the micrograph. Haematoxylin and eosin staining.
Denervated axolotl forelimbs stimulated to regenerate following the implantation of a single dorsal root ganglion (G). (A) An early three-digit regenerate (digits are numbered 1, 2 and 3) formed following the implantation of a ganglion 1 day after denervation (Series IV). The approximate location of the ganglion (G; not seen in this section) is just distal to the plane of amputation (midradius-ulna; dashed line). The bar represents 200 μ m. (B) A ganglion (G) implanted 4 days after denervation (Series IV) stimulated the growth of this palette-stage regenerate. Note the large accumulation of blastema cells distal to the ganglion which is near the plane of amputation (dashed line). The bar represents 175 gm. (C) An enlargement of half of the ganglion in B showing the abundant neuronal cell bodies, some of which are indicated by arrows. The bar represents 100 μ m. (D) A further enlargement of the ganglion in C to show the abundant neurones (arrows) found in these limbs. The bar represents 5 μ m. In C and D regeneration is directed toward the top of the micrograph. Haematoxylin and eosin staining.
Fig. 6 is representative of redenervated nonimplanted axolotl limbs and redenervated limbs with liver or muscle implants; all three of these control limb groups exhibited only pseudoblastemata typical of resorbing denervated larval limbs (Schotté & Butler, 1941; Olsen & Tassava, 1984). In axolotl limbs that were stimulated to regenerate by DRG implants, large numbers of blastema cells were present at the distal end of the limb (Fig. 5). The cellular accumulation obscuring DRG homografts in adult newt limbs was not observed in association with DRG xenografts in axolotl limbs (compare Figs 4C,D and 5C,D). With respect to the original plane of amputation, the position of the DRG in the distal stump varied somewhat from limb to limb (compare Figs 5 and 7), but whether a correlation existed between the final location of the DRG and the extent/frequency of regeneration could not be ascertained.
Histological section through a denervated control limb implanted with newt flank muscle. Note the small accumulation of cells indicative of a pseudoblastema (pb). Due to resorption following denervation, the original plane of amputation was distal to, but approximately parallel with, the dashed line. Evidence of continued resorption is seen as distal portions of the humerus (h) undergo histolysis. Bar represents 200 μ m. Haematoxylin and eosin staining.
Histological section through a denervated control limb implanted with newt flank muscle. Note the small accumulation of cells indicative of a pseudoblastema (pb). Due to resorption following denervation, the original plane of amputation was distal to, but approximately parallel with, the dashed line. Evidence of continued resorption is seen as distal portions of the humerus (h) undergo histolysis. Bar represents 200 μ m. Haematoxylin and eosin staining.
(A) A nonregenerating denervated limb that contained an implanted ganglion. Note the large accumulation of cartilage (c) at the end of the limb. (B) Another section of the limb in A which shows that the position of the ganglion (G) is a considerable distance proximal to the original plane of amputation (dashed line). The bar represents 200 μ m. Haematoxylin and eosin staining.
(A) A nonregenerating denervated limb that contained an implanted ganglion. Note the large accumulation of cartilage (c) at the end of the limb. (B) Another section of the limb in A which shows that the position of the ganglion (G) is a considerable distance proximal to the original plane of amputation (dashed line). The bar represents 200 μ m. Haematoxylin and eosin staining.
In newt limbs nerve fibre growth associated with the implanted DRG was not observed. In axolotl forelimb regenerates, staining revealed nerve fibre outgrowth from DRG in 82 % (28/34) of the limbs. In six regenerates nerve fibre growth from the DRG was not observed (Fig. 8B), in eighteen regenerates the ganglia produced only a few nerve fibres (Fig. 8C) and in ten cases a large number of nerve fibres coursed through the regenerate and directly into the apical epidermis (Fig. 8D). In nonregenerating DRG-implanted limbs, nerve fibre growth from the ganglia was observed in only two of fifteen cases and in both instances the fibres were restricted to the margins of the DRG implants (similar to that shown in Fig. 8B).
(A) A typical cone-stage regenerate on a denervated axolotl limb stimulated by an implanted dorsal root ganglion (G). Note the distal accumulation of blastema cells. The bar represents 250 μ m. Haematoxylin and eosin staining. The remaining photomicrographs are enlargements of silver-stained sections showing nerve fibre growth associated with the ganglia (which is located on the left of each micrograph). (B) This DRG supported the regeneration of the axolotl limb shown in Fig. 5B. Fibre growth was limited to within the margins of the ganglion and few fibres (if any) could be seen within the blastema. (C) The ganglion shown here is the same one shown in Fig. 8A. Note the numerous fibres that extend beyond the margins of the ganglion toward the regenerate. Nerve fibres were seen scattered throughout this regenerate. (D) An example of abundant nerve fibre growth seen from an implanted DRG. This dense accumulation of fibres eminating from the DRG coursed through the associated cone-stage regenerate (not shown). For the purpose of orientation the direction of regeneration in each of B, C and D is towards the right of the micrograph and the bar in each of these figures represents 100 μ m.
(A) A typical cone-stage regenerate on a denervated axolotl limb stimulated by an implanted dorsal root ganglion (G). Note the distal accumulation of blastema cells. The bar represents 250 μ m. Haematoxylin and eosin staining. The remaining photomicrographs are enlargements of silver-stained sections showing nerve fibre growth associated with the ganglia (which is located on the left of each micrograph). (B) This DRG supported the regeneration of the axolotl limb shown in Fig. 5B. Fibre growth was limited to within the margins of the ganglion and few fibres (if any) could be seen within the blastema. (C) The ganglion shown here is the same one shown in Fig. 8A. Note the numerous fibres that extend beyond the margins of the ganglion toward the regenerate. Nerve fibres were seen scattered throughout this regenerate. (D) An example of abundant nerve fibre growth seen from an implanted DRG. This dense accumulation of fibres eminating from the DRG coursed through the associated cone-stage regenerate (not shown). For the purpose of orientation the direction of regeneration in each of B, C and D is towards the right of the micrograph and the bar in each of these figures represents 100 μ m.
Discussion
DRG grafts provide the neurotrophic stimulation essential for forelimb regeneration in both adult newts and larval axolotls. Excision and immediate autografting of brachial ganglia (Kamrin & Singer, 1959) has been shown to stimulate continued regeneration of the adult newt limb. While this procedure eliminates the sensory supply, without redenervation it does not prevent motor nerves from regenerating into the limb and supplementing the neurotrophic stimulation offered by the grafted DRG. The present procedure of homografting DRG and repeatedly transecting the host brachial plexus demonstrated that DRG did stimulate forelimb regeneration independent of the host nerve supply. Furthermore, pituitary gland implants were ineffective stimulators of blastema outgrowth arguing against the view that pituitary secretions may be functionally identical to neurotrophic factors (Wallace, 1981).
In adult newts, the efficacy of DRG stimulation of regeneration is related to the number of blastema cells present at the time of denervation and the timing of DRG implantation with respect to denervation. With each increase in time prior to denervation and DRG grafting (1, 10 and 14 days), larger percentages (0, 34 and 57 %) of adult newt limbs regenerated, thus suggesting a correlation between DRG stimulation and the known increase in blastema cell numbers through this time period (Chalkley, 1954). As the interval between amputation and denervation was extended (to 14 days) in denervated control limbs, the development of small nerve-independent spikes (Singer & Craven, 1948) was sometimes observed, presumably due to the accumulation of a stable population of blastema cells. While denervation on day 10 resulted in a total inhibition of control limb regeneration, it is significant that even with the smaller population of blastema cells, implanted DRG nevertheless stimulated regeneration at this early nerve-dependent stage. Furthermore, the efficacy of stimulation by ganglia on day 14 was demonstrated by the increased size and morphological complexity of the regenerates on experimental limbs compared to the nerve-independent spikes observed on denervated control limbs.
The protocol of reducing residual neurotrophic activity by denervating for some period of time and subsequently implanting DRG to restimulate regeneration has not previously been investigated in vivo. Kamrin & Singer (1959) only used the maintenance strategy in their study which demonstrated that autoplastically grafted DRG promoted outgrowth of regenerates on newt forelimbs. Similarly, the maintenance strategy using cocultured DRG in vitro (whereby innervated regenerates were excised and immediately cocultured with DRG) has been shown to stimulate macromolecular synthesis (Vethamany-Globus et al. 1978) and mitosis (Globus & Vethamany-Globus, 1977; Tomlinson et al. 1981). However, based on data from a combined in vivo and in vitro study, Tomlinson et al. (1984) suggested that blastema cells may become refractory to neurotrophic stimulation following denervation. Regenerates that had been denervated for 48 h in vivo did not subsequently respond to DRG stimulation when explanted in vitro (i.e. restimulation). Other studies using extracts or purified molecules have only employed the restimulation strategy to investigate the neurotrophic stimulation of regeneration. Infusions of purified molecules (Mescher & Gospodarowicz, 1979; Carlone & Rathbone, 1985) or nerve extracts (Lebowitz & Singer, 1970; Singer et al. 1976; Jabaily & Singer, 1977; Carlone & Foret, 1979) 48 h after denervation stimulated only limited increases in biological activity and did not result in regenerate outgrowth (Wallace, 1981).
Results of the present investigation in which maintenance and restimulation strategies were compared are consistent with the view (Tomlinson et al. 1984) that, soon after denervation, adult newt blastemata lose responsiveness to restimulation by nerves or nerve extracts. As the length of the denervation time was increased prior to ganglia implantation, successively fewer newt limbs regenerated. DRG implanted before, or concomitant with, denervation stimulated regeneration, but their effectiveness was reduced after only a 24 h denervation time suggesting relatively rapid changes within the blastema. It is interesting to note that some parameters indicative of cell cycle activity (DNA synthesis, Singer & Caston, 1972; mitosis, Tomlinson et al. 1984) remain elevated for up to 48 h after denervation. In this regard, we can now investigate the DRG stimulation of regeneration of denervated adult newt limbs in relation to the size and proliferative status of the blastema.
The present study provides the first evidence that denervated larval axolotl limbs are also capable of regenerating in response to implanted DRG. Both maintenance and restimulation strategies were effective in promoting the regeneration of denervated axolotl limbs at a relatively high frequency apparently independent of the size of the blastema and timing of DRG implantation with respect to denervation. It is of interest to consider the timing of DRG implantation relative to amputation in axolotls with regard to the development of an in vivo bioassay. Between 4 and 7 days following simultaneous amputation and denervation there is a limited and transient burst of cell cycle activity (Maden, 1978) which is subsequently suppressed (Olsen et al. 1984; Barger & Tassava, 1985). Therefore DRG implanted concomitant with, or 4 days after, denervation (Series IV, 1 and 5 days after amputation, respectively) stimulated regeneration by maintaining cell cycle activity. In addition, DRG grafted concomitant with denervation 4 days after amputation (Series V) also maintained cycling activity and stimulated regeneration. By 8 days after amputation residual cell cycle activity was suppressed in those limbs that had been denervated for 4 (Series V) or 8 (Series IV) days (Tassava & McCullough, 1978; Maden, 1978) and therefore DRG implants presumably restimulated cell cycling to promote regeneration. Grafted dorsal root ganglia therefore are a complete source of factors necessary to restimulate regeneration-specific events in denervated axolotl limbs. The present DRG in vivo system can now be used to investigate short-term cell cycle events by both the maintenance and restimulation strategies and, in addition, it provides the opportunity to examine sustained growth and morphogenesis.
The ability of newt DRG to stimulate regeneration in denervated axolotl limbs reflects the fact that neurotrophic factors lack species specificity (Kamrin & Singer, 1959). Why then were differences in response to DRG observed between newts and axolotls? How can it be explained that DRG stimulated a higher frequency of regeneration in axolotl limbs after prolonged denervation and why was nerve fibre growth from the grafted ganglia more commonly observed in axolotl regenerates? At least part of the reason may involve the faster rate of regeneration (Tomlinson et al. 1985) and apparent immunological tolerance of the graft in axolotls. Evidence for immunological graft rejection was observed in adult newt limbs but not in larval axolotl limbs. Since both newts and axolotls are known to be capable of rejecting tissue grafts (reviewed by Wallace, 1981), the lack of such a response in the larval axolotl limb may be the consequence of the faster completion of regeneration, resulting in fixation for histology prior to the onset of a graft-rejection response.
Regardless of immunological complications, grafted DRG stimulated some regeneration in each species using both maintenance and restimulation strategies; however, while DRG were equally effective in both maintenance and restimulation strategies in axolotls, the restimulation strategy was considerably less effective than the maintenance strategy in stimulating the denervated adult newt limb to regenerate. Differing responses to DRG implants may also be related in part to different intrinsic tissue responses to neurotrophic stimulation. For example, the reinnervation of a previously denervated newt limb is not sufficient to stimulate regeneration (the limb must also be reamputated; Salley & Tassava, 1981), but in axolotls reinnervation is sufficient to stimulate regeneration (Schotté & Butler, 1949; Petrosky et al. 1980).
Utilization of the larval system as a reproducible regeneration-specific bioassay is a possibility worthy of further exploration. Outgrowth of a regenerate on a denervated limb, stimulated by the appropriate neurotrophic treatment, is a rigorous but ultimately essential assay for neurotrophic activity. Since the present studies show that DRG implants do stimulate blastema formation and growth, future experiments can be designed to examine the macromolecular and/or cell cycle events that occur shortly after implantation of DRG. Once the specific events influenced by grafted DRG have been defined it will be possible to determine if various DRG extracts and/or putative neurotrophic molecules influence the parameters in an identical manner.
ACKNOWLEDGEMENTS
This research was supported by NSF grant PCM 8315428 and USPHS grant NS-10165. Critical review of the manuscript by David J. Goldhamer is appreciated. We would also like to thank Donna E. Tomlinson for technical assistance and preparation of the manuscript.