A transgenic approach to identify thyroxine transporter-expressing structures in brain development.

Transporters are essential in thyroid hormone metabolism. Thyroxine (T4) is transported by solute carrier organic anion transporter 1c1 (SLCO1C1, OATP14) into the adult brain, where T4 is converted to 3,5,3'-triiodothyronine (T3). In adults, SLCO1C1 expression is found in two brain barrier structures: the blood-brain barrier (BBB) and choroid plexus. However, little is known about how T4 is transported in the developing brain, when the BBB is not yet completely formed. We employed bacterial artificial chromosome recombineering to generate transgenic mice carrying Cre recombinase in the Slco1c1 locus (Slco1c1-Cre mice). In Slco1c1-Cre mice Cre was expressed at the sites that have been previously reported for SLCO1C1 in adults. To trace Cre expression during development, we crossed Slco1c1-Cre transgenic mice with Rosa26 reporter mice. ß-galactosidase staining showed Cre activity in neurones of various brain structures, such as cortical layer 2/3 and the hippocampus, suggesting transient Slco1c1 expression during brain development. At embryonic day15, SLCO1C1 was expressed at the same site as TBR2, a marker of neuronal progenitors. Neurones that express SLCO1C1 during their development could be T4 sensitive. In support of this hypothesis, hypothyroxinaemia induced by propylthiouracil treatment of dams decreased the number of ß-galactosidase-positive neurones in cortical layer 2/3 of newborn Slco1c1-Cre/Rosa26 mice. In conclusion, by generating Slco1c1-Cre transgenic mice, we demonstrated that SLCO1C1 is expressed in the neuronal cell lineage during brain development. Expression of SLCO1C1 may underlie the extraordinary sensitivity of specific neuronal populations to hypothyroxinaemia.

Interleukin-2 gene therapy of chronic neuropathic pain.

Previous research has revealed an antinociceptive (analgesic) effect of interleukin-2 (IL-2) in central and peripheral nervous systems. Unfortunately IL-2 is very short-lived in vivo, so it is impractical to apply IL-2 for analgesia in clinic. This study was performed to evaluate the effect of intrathecal delivery of human IL-2 gene on rat chronic neuropathic pain induced by chronic constriction injury of the sciatic nerve. Human IL-2 cDNA was cloned into pcDNA3 containing a cytomegalovirus promoter. The paw-withdrawal latency induced by radiant heat was used to measure the pain threshold. The results showed that recombinant human IL-2 had a dose-dependent antinociceptive effect, but that this only lasted for 10-25 min. The pcDNA3-IL-2 or pcDNA3-IL-2/lipofectamine complex in contrast also showed dose-dependent antinociceptive effects, but these reached a peak at day 2-3 and were maintained for up to 6 days. Liposome-mediated pcDNA3-IL-2 produced a more powerful antinociceptive effect than pcDNA3-IL-2 alone. The paw-withdrawal latencies were not affected by control treatments such as vehicle, lipofectamine, pcDNA3, or pcDNA3-lipofectamine. In the experimental groups, human IL-2 mRNA was detected by reverse transcription-polymerase chain reaction in the lumbar spinal pia mater, dorsal root ganglion, sciatic nerve, and spinal dorsal horn, but not in gastrocnemius muscle. The expressed IL-2 profile detected by western blot coincided with its mRNA profile except it was present in the spinal dorsal horn at a higher level. Furthermore, human IL-2 assayed by enzyme-linked immunosorbent assay in cerebrospinal fluid could still be detected at day 6, but lower than day 3. The antinociceptive effect of pcDNA3-IL-2 could be blocked by naloxone, showing some relationship of the antinociceptive effect produced by IL-2 gene to the opioid receptors. It is hoped that the new delivery approach of a single intrathecal injection of the IL-2 gene described here may be of some practical use as a part of a gene therapy for treating neuropathic pain.