In Graves' disease, the overstimulation of the thyroid gland and hyperthyroidism are caused by autoantibodies directed against the TSH receptor (TSHR) that mimics the action of TSH. The establishment of an animal model is an important step to study the pathophysiology of autoimmune hyperthyroidism and for immunological analysis. In this study, we adopted the technique of electroporation (EP) for genetic immunization to achieve considerable enhancement of in vivo human TSHR (hTSHR) expression and efficient induction of hyperthyroidism in mice. In a preliminary study using beta-galactosidase (beta-gal) expression vectors, beta-gal introduced into the muscle by EP showed over 40-fold higher enzymatic activity than that introduced via previous direct gene transfer methods. The sustained hTSHR mRNA expression derived from cDNA transferred by EP was detectable in muscle tissue for at least 2 wk by RT-PCR. Based on these results, we induced hyperthyroidism via two expression vectors inserted with hTSHR or hTSHR289His cDNA. Consequently, 12.0-31.8% BALB/c mice immunized with hTSHR and 79.2-95.7% immunized with hTSHR289His showed high total T(4) levels due to the TSHR-stimulating antibody after three to four times repeated immunization by EP, and thyroid follicles of which were hyperplastic and had highly irregular epithelium. Moreover, TSHR-stimulating antibody surprisingly persisted more than 8 months after the last immunization. These results demonstrate that genetic immunization by in vivo EP is more efficient than previous procedures, and that it is useful for delineating the pathophysiology of Graves' disease.

Aims:To evaluate the ability of TAC-302, a cyclohexenoic fatty alcohol derivative, to enhance neurite outgrowth in cultured rat dorsal root ganglion (DRG) neurons, and the preventive effects of TAC-302 on bladder denervation-related storage and voiding dysfunctions in rats with bladder outlet obstruction (BOO). Methods: Rat DRG neurons were cultured in the presence of TAC-302. Cell numbers and neurite lengths were quantified after a 24 h culture. BOO was achieved by partial ligature of the proximal urethra in female rats. BOO rats were divided into three groups and orally treated with vehicle of 3 or 30 mg/kg TAC-302 twice a day for 4 weeks. Cystometry was performed under conscious conditions. Immunohistochemical staining using anti-PGP9.5 of the bladder muscle layer was performed, and the innervation area was scored. Results: TAC-302 significantly and dose-dependently increased neurite outgrowth in cultured DRG neurons. BOO rats showed a decreased innervation area in the urinary bladder compared to sham-operated rats. BOO-induced denervation of the urinary bladder was partially prevented by oral treatment with TAC-302. TAC-302 significantly reduced the frequency of non-voiding contraction (NVC) and residual urine volume (RUV) compared with the BOO vehicle group (P < 0.05). The innervation area score exhibited significant negative correlations with NVC and RUV, indicating that they increased according to the progression of denervation. Conclusions: Our data indicate that TAC-302 promotes neurite outgrowth in vitro. In addition, TAC-302 prevents BOO-induced bladder dysfunction in rats, and has a protective effect on bladder denervation. K E Y W O R D Sbladder denervation, bladder outlet obstruction, cystometry, detrusor overactivity, This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

TAC-302 improved storage and voiding dysfunctions by improving bladder denervation and detrusor underactivity even when the treatment was started after storage and voiding dysfunctions had already occurred.

Aims: Sensory information from the lower urinary tract (LUT) is conveyed to the spinal cord to trigger and co-ordinate micturition. However, it is not fully understood how spinal dorsal horn neurons are excited during the voiding reflex. In this study, we developed an in vivo technique allowing recording of superficial dorsal horn (SDH) neurons concurrent with intravesical pressure (IVP) during the micturition cycle in both normal and diabetic rats.Methods: Lumbosacral dorsal horn neuronal activity and IVP were recorded from urethane-anesthetized naive and streptozotocin (STZ)-induced diabetic rats. Saline was continuously perfused into the urinary bladder through a cannula to induce micturition. Results: We classified SDH neurons into bladder-and urethral-responsive neurons, based on their responsiveness during the voiding reflex. Bladderresponsive SDH neurons responded to the rapid increase in IVP at the start of voiding. In contrast, urethral-responsive SDH neuronal firing increased at the peak IVP and their firing lasted during the voiding phase (the high-frequency oscillations). Urethral-responsive SDH neurons were more sensitive to capsaicin, received C afferent fiber inputs, and were rarely detected in STZdiabetes rats. Administration of a cyclohexenoic long-chain fatty alcohol (TAC-302), which is reported to promote neurite outgrowth of peripheral nerves in STZ-diabetic rats, prevented the functional loss of spinal urethral response. Conclusions: Sensory information from the bladder and urethra is conveyed separately to different groups of SDH neurons. Functional loss of spinal urethral sensory information through unmyelinated C afferent fibers may contribute to diabetic bladder dysfunction. K E Y W O R D S C fiber, capsaicin, diabetic bladder dysfunction, dorsal root ganglion, sensory response, spinal neuronal activity