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Endocrinology Vol. 142, No. 12 5190-5197
Copyright © 2001 by The Endocrine Society

TRH-TSH-THYROID

Induction of Type 3 Iodothyronine Deiodinase by Nerve Injury in the Rat Peripheral Nervous System

Wen Wu Li, Claude Le Goascogne, Martine Ramaugé, Michael Schumacher, Michel Pierre and Françoise Courtin

INSERM, U-488, 94276 Le Kremlin-Bicêtre, France

Address all correspondence and requests for reprints to: Dr. Françoise Courtin, INSERM, U-488, 80 rue du Général Leclerc, 94276 Le Kremlin-Bicêtre Cedex, France. E-mail: courtin{at}kb.inserm.fr


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Thyroid hormones are essential for the development and repair of the peripheral nervous system. The type 2 deiodinase, which is responsible for the activation of T4 into T3, is induced in injured sciatic nerve. To obtain information on the type 3 deiodinase (D3) responsible for the degradation of thyroid hormones, we looked for its expression (mRNA and activity) in the sciatic nerve after injury. D3 was undetectable in the intact sciatic nerve of adult rats, but was rapidly and highly increased in the distal and proximal segments after nerve lesion. After cryolesion, D3 up-regulation disappeared after 3 d in the proximal segment, whereas it was sustained for 10 d in the distal segment, then declined to reach basal levels after 28 d, when functional recovery was completed. After a transsection preventing the nerve regeneration, up-regulation of D3 persisted up to 28 d at high levels in the distal segment. D3 was expressed in peripheral connective sheaths and in the internal endoneural compartment. D3 mRNA was inducible by 12-O-tetradecanoylphorbol-13-acetate in cultured fibroblasts or Schwann cells. In conclusion, induction of D3 in the peripheral nervous system after injury may play an important role during the regeneration process by adjusting intracellular T3 levels.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THYROID HORMONES (T3 and T4) are known to be important physiological regulators for the development and maturation of the central and peripheral nervous system (for review, see Refs. 1, 2, 3). Thyroid hormones also seem to be essential for peripheral nerve repair (for review, see Ref. 4). Indeed, T3 has been reported to accelerate neuromuscular reinnervation (5). Furthermore, peripheral nerve regeneration is accelerated by the ip injection of thyroid hormones (6, 7) and their local administration in a silicone chamber at the level of the transsected sciatic nerve (8). Finally, nuclear thyroid hormone receptors, which are believed to mediate thyroid hormone action, are expressed in the adult rat sciatic nerve after transsection (9).

The deiodination of thyroid hormones in selected extrathyroidal tissues is believed to play an important role in modulating thyroid hormone action. The type 2 deiodinase (D2) (see Ref. 10 for review) activates T4 into T3, the more active thyroid hormone, by 5'-deiodination. In the brain, for example, 80% of T3 is formed through local D2 activity (11, 12). The type 3 deiodinase (D3), which is responsible for the degradation of thyroid hormones, T4 and T3, by 5-deiodination, is also highly expressed in the brain (see Ref. 10 for review). Thyroid status affects the expression of cerebral D2 and D3, thus regulating the intracellular concentration of T3 (13, 14). In cultured astroglial cells, D2 and D3 are strongly induced by various factors, including cAMP (15, 16), hormones (17, 18, 19, 20, 21), neuromediators (15), growth factors (21, 22, 23), and drugs that activate PKC, such as phorbol esters (17, 21, 23). Inhibition of D2 activity by T4, rT3 (24), and, to a lesser extent, T3 (18) and induction of D3 by thyroid hormones in astroglial cells (18, 21) contribute to the effects of thyroid status on D2 and D3 expression in the brain.

Although thyroid hormone metabolism in the central nervous system is well documented, there was a complete lack of information for the peripheral nervous system (PNS) until our very recent demonstration of the induction of D2 in the peripheral connective sheaths after sciatic nerve injury (25). To obtain information related to D3 in the PNS, the expression of D3 mRNA and activity has been studied in the sciatic nerve of adult rats under normal conditions or after injury.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Male Sprague Dawley rats [Iffa Credo (l’Arbresle, France) and our own breeding laboratory] were housed in a 12-h light, 12-h dark cycle and allowed free access to water and standard laboratory animal diets. All experiments were carried out in accordance with French Decree A 94120 (1991) and associated guidelines of European Economic Community Directive 86/609/EEC (1986).

Surgical procedures for lesion of the sciatic nerve
Nine- to 10-wk-old rats were deeply anesthetized with 150 mg/kg ketamine hydrochloride (Imalgène, Rhône Mérieux, Paris, France) and 3 mg/kg acepromazine (Vetranquil, Sanofi Pharmaceuticals, Inc., France) ip. The right sciatic nerve was exposed, and two types of lesions, i.e. cryolesion and transsection, were performed at the level of the gluteus maximus muscle. To allow subsequent regeneration of axons into the distal portion, the nerve was cryolesioned. The nerve was submitted to six cycles of freezing-thawing using a copper cryode (1-mm diameter) precooled in liquid nitrogen (26). The extent of the lesion was about 3 mm. The cryolesion site was marked with a single epineural 10-0 monofilament nylon suture (Ethicon, Neuilly/Seine, France). To prevent regeneration of the axons into the distal portion, the nerve was transsected, and the distal stump was turned in the reverse direction and sutured to the epimysium. The skin incision was closed with wound clips. On postoperative d 1, 3, 6, 10, 15, 28, and 40, the rats were killed by decapitation, and the proximal and distal nerve segments and dorsal root ganglia (DRGs) corresponding to vertebral level L4/L6 were aseptically collected, immediately frozen in liquid nitrogen, and stored at -80 C until RNA extraction. The contralateral DRGs of some rats were also sampled. In sham-operated rats the sciatic nerve was simply exposed. In some experiments the peripheral nerve sheaths (epineurium and perineurium) were stripped off from the axial components (endoneurium and nerve fibers in intact nerves) under a stereomicroscope.

Schwann cell and fibroblast cultures from neonatal rat sciatic nerves
Purified Schwann cells were prepared as previously described (27). Briefly, cells from sciatic nerves of 4-d-old Sprague Dawley rats were dissociated with trypsin (0.25%; Bio Media, Boussens, France) and type 1 A collagenase (0.1%; C-9891, Sigma, St. Louis, MO) and cultured in DMEM (Bio Media) containing 10% FCS (Eurobio, Les Ulis, France), penicillin (100 IU/ml), and streptomycin (100 µg/ml) in 25-cm2 tissue culture flasks precoated with poly-L-lysine hydrobromide (molecular masses, >300 kDa; Sigma). Contaminating fibroblasts were partially eliminated by treating the cultures for 48 h with 10 µM of the antimitotic cytosine arabinoside (Sigma). Schwann cells were induced to proliferate in the presence of the mitogens forskolin (2 µg/ml) and insulin (5 µg/ml). After cell confluence was reached on about d 11, residual fibroblasts were eliminated by treatment with an antibody against the membrane protein Thy-1.1 and Low-Tox rabbit complement (Cedarlane, Hornby, Ontario, Canada; supplied by TEBU, Le Perray-en-Yvelines, France). Then aliquots of the cells were plated on poly-L-lysine-treated petri dishes in DMEM-FCS with insulin and forskolin (not less than 8 x 104 cells/plate). The medium was changed every 2–3 d until cells reached confluence.

Fibroblasts were prepared as previously described (28) by the same procedure as that used for Schwann cells, but without treatments with cytosine arabinoside, Thy-1.1, and complement. The cells were allowed to reach confluence before subculturing and replating them (not less than 2 x 104 cells/plate) onto uncoated petri dishes to avoid adhesion of Schwann cells. They were grown in 10 ml DMEM-FCS with insulin, but without forskolin, to prevent Schwann cell proliferation.

When cells reached confluence, DMEM-FCS was removed, and the cells were washed with a 1:1 mixture of DMEM and Ham’s F-12 medium supplemented with 5.2 g/liter glucose, 1.8 g/liter sodium bicarbonate, and antibiotics. The cells were then cultured for 1 additional d in DMEM-Ham’s F-12 supplemented with 30 nM sodium selenite, 10 µg/ml insulin, and 10 µg/ml transferrin, followed by 24 h in DMEM-Ham’s F-12 supplemented with 30 nM sodium selenite, 1 µM cortisol, and 10 µg/ml transferrin.

RNA preparation
Frozen nerves and DRGs were ground into powder in a mortar precooled with liquid nitrogen, whereas cultured cells were lysed with lysis buffer RLT (QIAGEN, Courtaboeuf, Germany). Then total RNAs were extracted using TRIzol reagent (Life Technologies, Inc., Grand Island, NY) for tissue powder or the RNeasy Mini Kit (QIAGEN) for cultured cells according to the instructions of the manufacturers and were quantified by measurement of 260 nm absorption.

Semiquantitative RT-PCR
RT was performed during 1-h incubation at 42 C with 1 µg total RNA as template for each sample in a 20-µl reaction volume of reverse transcriptase buffer containing 10 mM dithiothreitol, 0.5 mM dNTPs mix, and 200 ng random primers, 20 U ribonuclease inhibitor, and 200 U Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc.). An appropriate volume of the same RT (1 µl, corresponding to ~50 ng cDNA) was used in subsequent D3 and S26 ribosomal protein PCR experiments. The PCR reaction was performed in 50 µl Expand High Fidelity buffer containing 1.5 mM MgCl2, 0.2 mM dNTPs, 0.1 µCi [{alpha}-32P]dCTP (Amersham Pharmacia Biotech, Little Chalfont, UK), 0.4 µM sense and antisense primers, and 2.6 U Expand High Fidelity PCR System (Roche, Mannheim, Germany). The oligonucleotide primers were derived from the coding regions of rat D3 cDNA (29) and rat S26 ribosomal protein cDNA, which were expected to give 386- and 261-bp products, respectively. Primers for D3 were: sense, 5'-CCC TGC TGC TTC ACT CTC TG-3'; and antisense, 5'-GGT CCC TTG TGC GTA GTC GA- 3'. Primers for S26 were: sense, 5'-GTG CGT GCC CAA GGA TAA GG-3'; and antisense, 5'-ATG GGC TTT GGT GGA GGT CG-3'. The conditions of amplification for D3 were 2 min at 94 C, followed by 30 cycles of denaturation (94 C, 1 min), annealing (64 C, 1 min), and extension (72 C, 1 min), followed by a final extension at 72 C for 2 min. The same conditions were used for S26, except for 25 cycles. Amplification products were separated on a 0.8% agarose gel and then transferred to a positively charged nylon membrane (Roche), exposed to Kodak x-ray film (Rochester, NY). The quantity of PCR product formed, which was evaluated by the quantity of incorporated radiolabeled nucleotide using an Instant Imager (Packard Instrument Co., Meridian, CT), increased in a linear manner as a function of the cDNA amount obtained from RT until 100 ng (see Fig. 1BGo) for both D3 and S26 ribosomal protein PCR. Reactions without template cDNA or with RNA extracts without RT were run as negative controls. A reaction with D3 cDNA obtained by RT from newborn Sprague Dawley rat astrocytes in primary culture was run as a positive control in each experiment.



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  		Figure 1. D3 mRNA expression in the sciatic nerve. The right sciatic nerve of rats was injured by freezing as described in Materials and Methods. One day after cryolesion, the distal segment of the nerve was removed, and total RNA was extracted. D3 mRNA levels were assessed with 1 µl RT, i.e. 50 ng cDNA, by RT-PCR assays. In this and the following figures, PCR for S26 ribosomal protein with the same RT were used as an internal standard, and RT-PCR from neonatal rat brain astrocytes in primary culture was run as a positive control (lane A). A, D3 mRNA levels in the sciatic nerve from five control rats (lanes 1–5) and in the distal segment of the injured nerve from five animals 1 d after a cryolesion (lanes 6–10). B, Semiquantitative RT-PCR validation. Various amounts of cDNA obtained by RT from control ({square}, D3; {blacksquare}, S26) or cryolesioned sciatic nerves ({circ}, D3; {bullet}, S26) were amplified by PCR. Quantifications (counts per min) were performed by Instant Imager analysis. C, Histogram showing D3 mRNA expression evaluated by the ratio of D3 to S26 PCR products (cpm incorporated in D3-PCR products/cpm incorporated in S26-PCR products x 100) for control nerves ({square}) and the distal segments of injured nerves ({blacksquare}). Results are the mean ± SD. ***, P < 0.001 vs. control value (n = 5). D, In another experiment, D3 mRNA levels were assessed in nerves from control (lanes 1–4) and sham-operated (lanes 5 and 6) rats and in the distal segment of injured (lane 7) rats.

 
Nucleotide sequencing
The 386-bp product derived from the RT-PCR was purified after gel electrophoresis on a 1% agarose gel using the QIAquick gel extraction kit (QIAGEN). This DNA fragment was then sequenced on both strands using the same primers as those used for the PCR amplification and an automated sequencing system with fluorescent dye terminators (PE Applied Biosystems, Foster City, CA).

D2 and D3 activity assays
The total nerves, the peripheral nerve sheaths (external compartment), or the internal compartment of sciatic nerves, either intact or after cryolesion or section, were homogenized with a glass potter at 4 C in 150 µl (for nerve sheaths), 300 µl (for internal compartment), or 450 µl (for total nerve) 80 mM ß-glycerophosphate buffer, pH 7.4, containing 15 mM MgCl2, 20 mM EGTA, protease inhibitors (1 mM phenylmethylsulfonylfluoride, 50 µg/ml aprotinin, 4 µg/ml leupeptin, 10 µg/ml antipain, 1 mM trypsin inhibitor, 1 mM benzamidine, and 10 µg/ml pepstatin), and 1 mM of the phosphatase inhibitor orthovanadate (Na3VO4). Aliquots of homogenates were immediately frozen and kept at -80 C. The protein concentration of tissue homogenates was determined by the method of Bradford (30) using BSA as the standard. For D2 activity assays, homogenates with the same amount of protein (40 µg) were incubated at 37 C for 60 min in a final volume of 80 µl containing 20 mM HEPES buffer (pH 7.4), 20 mM dithiothreitol, 50 nM T3, and 1 nM [125I]T4 (Amersham Pharmacia Biotech). Reactions were stopped by adding 10 µl NH4OH (10 M) containing 10 µM T3 and 10 µM T4. The [125I]T3 produced was separated from [125I]T4 by descending paper chromatography (31). Then the radioactive products were counted for determination of D2 activity, expressed as femtomoles of T3 per min/mg proteins. Deiodination was linear with respect to both protein concentration and incubation time. For D3 activity assays, homogenates with the same amount of protein (40 µg) were incubated at 37 C for 60 min in a final volume of 80 µl containing 20 mM HEPES buffer (pH 7.4), 20 mM dithiothreitol, and 5 nM [125I]T3 (Amersham Pharmacia Biotech). Reactions were stopped by adding 10 µl NH4OH (10 M) containing 10 µM T3 and 10 µM T4. The [125I]3,3'-diiodothyronine (3,3'-T2) produced was separated from [125I]T3 by descending paper chromatography (31). Then the radioactive products were counted for determination of D3 activity, expressed as femtomoles of 3,3'-T2 per min/mg proteins. Deiodination was linear with respect to both protein concentration and incubation time.

Statistical analysis
Statistical differences between two groups were determined using Mann-Whitney tests and were considered significant at P < 0.05. For statistical analysis concerning mRNA evaluated by the ratio of D3/S26 PCR products, data from each experiment were expressed as the fold increase over the mean control value. The SD of the control values was calculated on the basis of the control values obtained in the same PCRs.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of sciatic nerve lesion on D3 mRNA expression
Semiquantitative RT-PCR were carried out on total RNA extracted from sciatic nerves. In parallel with PCRs for D3, PCRs for the S26 ribosomal protein were performed as an internal standard (Fig. 1AGo). Figure 1BGo illustrates the validity of the semiquantitative RT-PCR method. RTs were first performed with 1 µg total RNA from control and injured nerves. All total RNAs were assumed to be transcribed into cDNAs. Thereafter, PCRs for D3 and S26 were assayed in half-dilution series (200, 100, 50, 25, and 12.5 ng cDNA). Quantification of PCR product accumulation for D3 shows a linear increase with the amount of cDNA until 200 ng for intact and injured nerves. In the case of S26 ribosomal protein, accumulation of PCR products was linear up to 100 ng cDNA. Evaluation of D3 mRNA accumulation by the ratio of D3/S26 PCR products was unchanged up to 100 ng cDNA (not shown). In Fig. 1AGo and for all subsequent PCRs, a level of 50 ng cDNA was used for D3 and S26 PCR, allowing evaluation of D3 mRNA accumulation.

In the intact sciatic nerve of normal adult rats, D3 mRNA was constitutively expressed at low levels (Fig. 1Go, A and C). A cryolesion of the sciatic nerve induced a 3-fold increase in D3 mRNA expression in the distal segment of the nerve 1 d after the lesion. In the sham-operated rats, levels of D3 mRNA remained comparable to those observed in normal intact animals (Fig. 1DGo), indicating that the accumulation of D3 mRNA is really due to the lesion of the nerve and is not a consequence of the surgical stress. The unilateral sciatic nerve injury resulted in an early response, which was already apparent 4 h after cryolesion (Figs. 2Go and 5Go). During the first 24 h following the injury, D3 mRNA was increased not only in the nerve segment located distally to the lesion site, but also in the proximal segment. Individual variations in the amplitude of D3 mRNA were observed in the proximal and distal stumps.



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  		Figure 2. Sciatic nerve injury induces a rapid D3 mRNA response. A, D3 mRNA levels were followed by RT-PCR analysis in control rat nerve (C) and 4 h (2 rats), 7 h, 12 h, and 24 h (3 rats) after a cryolesion in the proximal (P) and distal (D) nerve segments. B, Evolution of D3 mRNA expression evaluated by the ratio of D3/S26 PCR products (mean ± SD at 4 and 24 h) in proximal ({circ}) and distal ({blacksquare}) segments after cryolesion.

 


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  		Figure 5. Time course of the increase in D3 mRNA expression in the sciatic nerve after lesion. A, D3 mRNAs levels were followed by RT-PCR at the different indicated times and were evaluated by the fold increase in the ratio of D3 to S26 PCR products over the values of intact sciatic nerves. A, After cryolesion. {square}, Intact sciatic nerves (n = 5); {blacksquare}, distal segment (4 and 7–12 h, n = 3; 1–2 d, n = 9; 3–10 d, n = 6; 15 and 28–40 d, n = 3); , proximal segments (4 and 7–12 h, n = 3; 1–2 d, n = 5; 3–10 d, n = 4; 15 and 28–40 d, n = 3). B, After section. {square}, Intact sciatic nerves (n = 5); {blacksquare}, distal segment (1 d, n = 3; 3–6 d, n = 4; 15–28 d, n = 5); , proximal segment (1 d, n = 3; 3–6 d; n = 4; 15–28 d, n = 5). Results are the mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (lesioned vs. control). a, P < 0.01; §, P < 0.001 (distal vs. proximal segment).

 
To verify the identity of the D3 transcripts, the 386-bp PCR product was purified and sequenced on both strands. The sequence of this PCR product was identical to that of D3 in rat placenta or neonatal skin (29).

Comparison of D3 expression after section or cryolesion of sciatic nerve
Local freezing of the sciatic nerve causes paralysis of the denervated muscles, which is only transient, as regrowth of severed axons is accomplished in a satisfactory way after Wallerian degeneration distally to the lesion site. The reason is that the continuity of the peripheral connective tissue sheaths, capillaries, and basal lamina tubes formed by Schwann cells at the periphery of nerve fibers is not disrupted by local deep-freezing injury, providing an appropriate environment for regeneration. On the other hand, transsection was performed with suture to prevent regenerating axons to reach the distal stump and to reinnervate the target muscles.

The time course of D3 expression was followed in the proximal and distal nerve segments after both types of lesion. The cryolesion elicited a rapid and strong induction of D3 mRNA for the first 48 h in the distal stump, followed by a plateau level until d 10 and thereafter a decline to the levels measured in the intact control nerves (Figs. 3Go and 5AGo). The increase in D3 mRNA was also observed in the proximal nerve segments during the first 2 d after cryolesion at a lower level than in the distal stump and for a shorter duration, as D3 mRNA had almost returned to basal levels after 3 d. In the first days following transsection, D3 mRNA expression was potently induced in both proximal and distal stumps (Figs. 4Go and 5BGo). This D3 mRNA accumulation was followed after 6 d by a decline to values near the control levels in the proximal stump, whereas the amount of D3 mRNA did not return to the basal level in the distal segment and remained elevated, even 4 wk posttrauma.



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  		Figure 3. Time course of the increase in D3 mRNA expression in the sciatic nerve after cryolesion. A, D3 mRNA levels were followed by RT-PCR analysis in intact sciatic nerves (C) and in the proximal (P) and distal (D) segments from 1–40 d post injury. B, Evolution of the expression of D3 mRNA evaluated by the ratio of D3/S26 PCR products in the proximal ({circ}) and distal ({blacksquare}) segments after cryolesion. This typical experiment reflects three independent experiments.

 


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  		Figure 4. Time course of the increase in D3 mRNA expression in the sciatic nerve after transsection. The right sciatic nerve was sectioned, and the formation of connections between the proximal and distal segments was prevented as described in Materials and Methods. A, D3 mRNA levels were followed by RT-PCR analysis in intact sciatic nerves (C) and in the proximal (P) and distal (D) segments from 1–28 d postinjury. B, Evolution of D3 mRNA expression evaluated by the ratio of D3 to S26 PCR products (mean ± SD at 24 h) in the proximal ({circ}) and distal ({blacksquare}) segments after section. This typical experiment reflects three independent experiments.

 
D3 activities were also measured in homogenates of intact nerves from normal rats and in the proximal and distal nerve segments after section or cryolesion. No activity could be detected in intact nerves (Fig. 6Go). After cryolesion, D3 activity was sharply increased by d 1 and decreased thereafter in a time course corresponding to that of D3 mRNA expression. In the proximal segment, D3 expression had almost disappeared after 3 d. After transsection, D3 activity remained at a high level in the distal segment at 3 wk and at a lower, but significantly increased, level in the proximal segment, in agreement with D3 mRNA measurements. These results show that D3 mRNA accumulation elicited by nerve lesion is effectively followed by enzyme expression. D3 activity is highly expressed in sciatic nerve after lesion. The maximal values approximate 10 fmol 3,3'-T2/min·mg protein, about 3-fold higher than the values obtained in adult frontal cerebral cortex homogenates (32), 10-fold higher than those in adult brain homogenates, similar to those in placenta on d 21 of gestation, and 2-fold higher than those in neonatal skin (33). However, they are 10-fold less than D3 activity measured in embryonal implantation site on d 9 (33), which is considered the highest level of D3 activity observed to date.



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  		Figure 6. Induction of D3 activity in the rat sciatic nerve by injuries. D3 activities were measured in homogenates of intact sciatic nerves (control; n = 11) or at the times indicated after lesion of the distal ({blacksquare}; 1 d after cryolesion, n = 4; 2–3 d after cryolesion, n = 13; 6–15 d after cryolesion, n = 7; 18–23 d after section, n = 17) or proximal (; 1 d after cryolesion, n = 4; 2–3 d after cryolesion, n = 8; 6–15 d after cryolesion, n = 7; 18–23 d after section, n = 12) segments of injured nerves. Results are the mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (lesioned vs. control). a, P < 0.01; §, P < 0.001 (distal vs. proximal segment).

 
D3 mRNA expression in DRGs after sciatic nerve lesion
Both cryolesion and section of the sciatic nerve induced a rapid increase in D3 mRNA, peaking at 1 d, in the L4/L6 ipsilateral DRGs (Fig. 7AGo). The response was more transient after cryolesion (not shown) than after section, persisting for 1 wk in the latter condition. Variations in D3 mRNA were not apparent in the contralateral sciatic nerve (not shown) or DRGs (Fig. 7BGo) after unilateral nerve injury.



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  		Figure 7. D3 mRNA expression in DRG after sciatic nerve injury. A, D3 mRNA levels were assessed by RT-PCR analysis in L4/L6 DRGs of control rats (C), and in the ipsilateral (IL) or contralateral (CL) L4/L6 DRGs 1 d after cryolesion or 1, 3, 6, and 15 d after section. For each sample, L4/L6 DRGs were pooled from three rats. B, Histogram showing D3 mRNA expression evaluated by ratios of D3/S26 PCR products in DRGs from control rats ({square}) and in ipsilateral ({blacksquare}) and contralateral () DRGs after cryolesion or section.

 
Localization of D3 expression
Schematically, a nerve is composed of a central area, comprising the nerve fibers (axons and Schwann cells) embedded in the endoneurium, and a peripheral zone of connective tissue sheaths, the epineurium and the perineurium, made from fibroblasts and collagen fibrils. In an attempt to determine the localization of D3, the peripheral connective nerve sheaths were stripped off from the central components of intact nerves and of the proximal and distal segments 3 wk after section or 6 d after cryolesion. RT-PCR analysis revealed the high expression of D3 mRNA in both the peripheral sheaths and the central area (Fig. 8AGo) in the distal stump, suggesting localization in fibroblasts and Schwann cells. In the proximal segment, D3 mRNA expression was very low 6 d after cryolesion in both external and internal compartments. D3 mRNA expression levels were also particularly low in the internal compartment of the proximal segment 3 wk after section, whereas D3 mRNA accumulation remained a little more sustained in the external segment. In vitro experiments performed with Schwann cells or fibroblasts isolated from neonatal sciatic nerves revealed that D3 mRNA was constitutively expressed in both cell types (Fig. 8BGo). T3 did not significantly affect D3 mRNA in fibroblasts, whereas 100 nM 12-O-tetradecanoylphorbol-13-acetate (TPA), a phorbol ester that activates PKC, potently induced D3 mRNA accumulation [control, 1 ± 0.12 (n = 5); T3-treated, 1.2 ± 0.2 (n = 4; P = NS); TPA-treated, 4.5 ± 1 (n = 3; P < 0.05, TPA-treated vs. control)]. T3 promoted a significant accumulation of D3 mRNA, and TPA potently induced D3 mRNA accumulation in Schwann cells [control, 1 ± 0.08 (n = 5); T3-treated, 1.4 ± 0.14 (n = 4; P < 0.05, T3-treated vs. control); TPA-treated, 3.6 ± 2.0 (n = 3; P < 0.05, TPA-treated vs. control)].



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  		Figure 8. Localization of D3 mRNA. A, The external nerve sheaths were separated from the internal compartment in control and lesioned sciatic nerves of adult rats (see Materials and Methods). D3 mRNA levels were followed by RT-PCR in the external (Ext) and internal (Int) compartments of intact sciatic nerve (C) and in the distal (D) and proximal (P) nerve segments 21 d after section or 6 d after cryolesion. For each sample, external and internal compartments were pooled from two rats. D3 mRNA levels were also followed 6 d after cryolesion in the proximal and distal segments of sciatic nerves without separation of the external and internal compartments (Total). B, Cell cultures from neonatal rat sciatic nerves. Purified Schwann cells and fibroblasts were cultured and maintained for the last 48 h in a chemically defined medium (see Materials and Methods). D3 mRNA levels were followed by RT-PCR in dishes treated for 24 h with 10 nM T3 or for 8 h with 100 nM TPA.

 
Measurements of D3 activity confirmed the RT-PCR localization of D3 expression. D3 activity was present at high levels in both the external connective tissue sheaths and the internal compartment (Fig. 9Go) of the distal nerve segment 2 d after cryolesion or 3 wk after section. After the initial increase in D3 activity observed in the proximal stump 1 d after cryolesion (Fig. 6Go), D3 activity appeared low in both internal and external compartments of the proximal segment at 2 d. Three weeks after section, D3 activity was undetectable in the internal compartment and at a low, but significantly increased, value in the external compartment of the proximal stump. In comparison, D2 activity was induced by lesion only in the external connective tissue sheaths, as previously shown (25). Two days after cryolesion, D2 activity was induced mostly in the distal nerve segment, whereas 3 wk after section, it was induced in both proximal and distal segments. The maximal value of D2 activity observed (0.4 fmol T3/min·mg protein) was about 2-fold lower than that obtained in frontal cerebral cortex homogenates (32), 4-fold higher than that obtained in total brain homogenates, and of the same order as that in brown adipose tissue or neonatal skin (33).



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  		Figure 9. Induction of D3 activity in external and internal compartments of the rat sciatic nerve by injuries; comparison with the induction of D2 activity. D3 activities were measured in homogenates of the external nerve sheaths (Ext; A) and the internal compartment (Int; B) prepared from intact sciatic nerves ({square}; n = 10) or from the distal ({blacksquare}; n = 5) and proximal (; n = 5) segments of injured nerves 2 d after cryolesion or 21 d after section. D2 activities were measured in homogenates of external nerve sheaths (C) and of the internal compartment (D) from intact sciatic nerves ({square}; n = 5) or from the distal ({blacksquare}; n = 10) or proximal (; n = 5) segments of injured nerves 2 d after cryolesion or 21 d after section. Results are the mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (lesioned vs. control). b, P < 0.05; a, P < 0.01; §, P < 0.001 (distal vs. proximal segment).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study shows for the first time the expression of D3 in the PNS. D3 expression is highly induced in the sciatic nerve after lesion, and thus this is also the first demonstration of D3 induction after injury. As a consequence, the PNS appears to have its own system for the local degradation of T3, which is inducible by lesion. Induction of D3 after injury may play an important role during the regeneration process to adjust intracellular T3 levels to the timing required for thyroid hormone action.

In the intact sciatic nerve of the rat, D3 expression is undetectable during development (our unpublished observations) and in the adult. D3 mRNA is strongly up-regulated in both the distal and proximal stumps 24 h after the two types of lesion performed, i.e. cryolesion and section. An increase in D3 mRNA expression is also observed in DRGs corresponding to the roots of the injured sciatic nerve. D3 induction is an early event after cryolesion, with D3 mRNA accumulation measured as early as 4 h and D3 activity strongly increased at 24 h. The signals that induce D3 in the sciatic nerve after injury remain to be determined. The ERK pathway has been recognized as an efficient inducer of D3 in astrocytes (21). Fibroblast growth factor-2, which induces D3 in astrocyte cultures (23) and is up-regulated in the sciatic nerve and DRGs as early as 1 d after lesion (34), can be a candidate among many factors up-regulated after nerve injury (for review, see Ref. 35). Early induction of D3 may also be controlled by signaling pathways activated in response to injury, as recently reported in the brain (36, 37).

After cryolesion, a type of injury that allows axonal regeneration, expression of D3 in the distal segment declines after a few days and completely disappears at the time of functional recovery, i.e. 28 d. The decline in D3 expression is more rapid in the proximal segment, where D3 is mostly absent after 3 d. Differences in the time course of D3 expression probably depend on precedence of regeneration processes in the proximal segment over those in the distal segment. After nerve transsection, a condition where no nerve repair or functional reinnervation takes place, the induction of D3 is sustained throughout the experiment, i.e. 28 d, in the distal stump. The high increase in D3 mRNA expression is transient in the proximal stump after section, but lasts for a longer period than after cryolesion, and a small D3 expression persists in this part, where nerve sprouting takes place.

D3 is expressed in the peripheral connective tissue sheaths of the distal stump of lesioned nerves. The connective tissue sheaths around peripheral nerves contain fibroblasts in the epineurium and perineurium that contribute to the blood-nerve barrier. D3 is also expressed in the endoneural inner compartment of the distal stump of lesioned nerves. One day after lesion, the inner compartment of the distal segment of injured nerves contains Schwann cells, fibroblasts, a few resident macrophages, and axons that have not yet undergone Wallerian degeneration (38). The recruitment of new macrophages is delayed on d 2 after lesion, allowing them to be discarded for D3 localization 1 d after lesion. A neuronal localization is also excluded in the distal stump physically separated from the neuronal cell bodies, although a contribution of neuronal expression is possible in the proximal stump and DRGs following injury. D3 has been shown to be expressed in neurons (39, 40) and astrocytes from central nervous system (23) and also in fibroblasts of Xenopus laevis tail (41). A report of a case of hemangioma also suggests possible localization in endothelial cells (42). Fibroblasts and Schwann cells are likely to express D3, as suggested by D3 mRNA accumulation in cultured fibroblasts or Schwann cells after stimulation by TPA, a phorbol ester increasing D3 activity in astrocytes through activation of PKC (21). The precise localization of D3 will require further studies.

Expression of D3 in the nerve after injury seems to partly coincide with the expression of nuclear TR (9). Indeed, there is no or low expression of TR in the intact sciatic nerve of adult rats, whereas TR are highly expressed for at least 2 wk (43) after sciatic nerve transsection, mainly in Schwann cells of the distal stump. The colocalization of D3 and TR in Schwann cells is possible, as D3 mRNA is expressed in cultured Schwann cells. Localization of D3 in endothelial cells of endoneural capillaries would also reduce the T3 content in Schwann cells, as a case of hemangioma with high D3 has been reported to promote hypothyroidism (42). High levels of D3 after cryolesion mean that degradation of T3 and T4 in the first 10 d of peripheral nerve regeneration would reduce thyroid hormone action. In the absence of thyroid hormone, TR are able to bind to their response sequences and to mediate gene silencing (see Ref. 44 for review). The role of D3 may thus be to delay myelination, possibly controlled by T3, as the myelin basic protein gene contains a functional thyroid response element in its promoter (45). The down-regulation of D3 after 10 d would allow T3 to reach its targets and promote myelination, which begins at this time after cryolesion (46). Interestingly, the expression of myelin basic protein mRNA begins to increase 7 d after a crush injury and is maximal at 21 d (47). In X. laevis metamorphosis, a decline of D3 in the tail permits the expression of delayed genes under T3 control (48). The origin of T3 in the nerve could be from the plasmatic T3 pool as well as from the local 5'-deiodination of T4 into T3, as D2, which is able to convert T4 into T3, is induced in connective nerve sheaths after sciatic nerve injury (25). Thus, D3 will contribute with D2 to the fine-tuning of permissive thyroid hormone action. In the proximal stump, which presents nerve sprouting, and the corresponding DRGs, increased D2 (25) is associated with low D3 3 wk after section. These conditions favor T3 accumulation, which may promote axon growth, as shown in vitro (49) and in vivo (8). After section, the distal stump contains undifferentiated dividing Schwann cells and exhibits a high D3 expression, raising the question of the role of T3 in regulating their multiplication and differentiation. T3 stops multiplication and promotes differentiation of oligodendrocyte precursor cells (50). Finally, understanding of the roles of T3 and D3 in nerve repair will require manipulation of D3 expression in the endoneural compartment.


   Acknowledgments
 
We thank D. Goudou for sequencing, L. Outin for image manipulation, and F. Robert for fruitful discussion.


   Footnotes
 
This work was supported in part by the Association pour la Recherche contre le Cancer (to M.P.) and the Centre National de la Recherche Scientifique. W.W.L. is a recipient of the Société de Secours des Amis des Sciences.

Abbreviations: D2, Type 2 deiodinase; D3, type 3 deiodinase; DRG, dorsal root ganglia; PNS, peripheral nervous system; 3,3'-T2, 3,3'- diiodothyronine; TPA, 12-O-tetradecanoylphorbol-13-acetate.

Received June 4, 2001.

Accepted for publication August 29, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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