Responsiveness of lumbosacral superficial dorsal horn neurons during the voiding reflex and functional loss of spinal urethral-responsive neurons in streptozotocin-induced diabetic rats.

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. Bladder-responsive 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 STZ-diabetes 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.

TAC-302 promotes neurite outgrowth of isolated peripheral neurons and prevents bladder denervation related bladder dysfunctions following bladder outlet obstruction in rats.

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.

Therapeutic effect of TAC-302, a cyclohexenoic fatty alcohol derivative, on bladder denervation-related storage and voiding dysfunctions in rats.

AIMS: To evaluate the therapeutic effect of TAC-302, a cyclohexenoic fatty alcohol derivative, on bladder denervation-related storage and voiding dysfunctions in rats with bladder outlet obstruction (BOO). METHODS: BOO was achieved by partial ligature of the proximal urethra in female rats. Two weeks later, BOO rats were divided into two groups and treated orally with vehicle or 10 mg/kg TAC-302 twice a day for 4 weeks. Urodynamic and immunohistochemical evaluation of the bladder muscle layer was performed. In another study, the BOO rats were treated with intravenous tamsulosin at cystometry. The detrusor contractility in each group was evaluated using the modified Shafer's nomogram. RESULTS: Two weeks after BOO, the rats showed significant increases in non-voiding contraction (NVCs) and residual urine volume (RUV) compared to the sham group. Moreover, 6 weeks after BOO, BOO vehicle rats showed significant increases in NVCs and RUV and decreases in detrusor contractility and in the nerve fiber density in the urinary bladder compared to the sham group. BOO-induced denervation of the urinary bladder was partially improved by oral treatment with TAC-302. Oral treatment with TAC-302 significantly reduced the amplitude and frequency of NVCs (P < 0.05) and increased detrusor contractility and tended to reduce RUV compared with the BOO vehicle group. In contrast, the intravenous administration of tamsulosin significantly reduced the frequency of NVCs, but not RUV. CONCLUSIONS: 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.

An improved Graves' disease model established by using in vivo electroporation exhibited long-term immunity to hyperthyroidism in BALB/c mice.

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.