Cellular and molecular mechanisms underlying functional plasticity of autonomic neurons in a diabetic rat model
당뇨병 쥐 모델의 자율신경 기능가소성의 세포 및 분자적 기전
Dept. of Medicine/박사
Diabetic peripheral neuropathy (DPN) is the most common complication of diabetic mellitus (DM) that can cause significant morbidity and mortality, and is found in >60% of patients over the course of their disease. DPN is characterized by diffuse or focal damages to peripheral somatic or autonomic nerve fibers which produce dysfunctions of skin, muscles, and visceral organs. The primary risk factor is hyperglycemia which activates multiple biochemical pathways including AGE, polyol, hexosamine, PKC, and PARP pathways to result in cell dysfunctions and death. Generally, DPN can be divided into sensory motor neuropathy and autonomic neuropathy. The latter is further classified as cardiaovascular, gastrointestinal, and genitourinary autonomic neuropathies. Among them, genitourinary autonomic neuropathy has been poorly understood in terms of its cellular and molecular mechanism. The major clinical symptoms of genitourinary autonomic neuropathy are bladder and sexual dysfunctions. Major pelvic ganglion (MPG), located on the lateral surfaces of the prostate gland in rat provides autonomic innervation to the distal colon and urogenital organs including the urinary bladder, the prostate, and the penis. Among autonomic ganglia, MPG is very unique since sympathetic and parasympathetic neurons are colocalized in the same ganglion capsule. Thus, MPG is critical for autonomic reflexes such as micturition and penile erection together with visceral sensory dorsal root ganglion (DRG). To date, it is little known whether DM affects functions of MPG and DRG neurons. Accordingly, I hypothesized that DM causes plastic changes of MPG and DRG neuronal functions by altering expression of certain types of ion channels, which may contribute to autonomic genitourinary dysfunctions. Thus, the purposes of the present study were to test whether excitability of MPG and DRG neurons is altered in
an experimantal diabetic rat model, and to define the molecular and cellular mechanisms underlying DM-induced changes in excitability.Experimental DM was induced by injection of streptozocin (STZ, 60 mg/kg, i.p.) into S/D rats (8 week-old). After three days, the levels of blood glucose in control and STZ-injected rats were measured. STZ-injected rats with hyperglycemia (>300 mg/dl) were divided into two groups (STZ and STZ+insluin). Insulin (10 IU) was injected into diabetic rats once per day. After 8 weeks, development of diabetic neuropathy in the STZ group was assessed by measuring motor nerve conduction velocity (MNCV) in sciatic nerves, myelin area, bladder micturition pattern, and intracarvernous pressure (ICP). As results, motor and autonomic neuropathies were found to be developed in the STZ group. H & E staining revealed the enlarged bladder with hypetropied urothelium in the STZ group. DM significantly decreased testosterone level while increased corticosterone, a stress hormone. Interestingly, DM was found to increase serum and tissue oxidative stress as the malondialdehyde level was measured. Under the gramicidine-perforated configuration of the patch-clamp techniques, spike firing was recorded in MPG and capsaicin-sensitive C-fiber DRG neurons (L6-S1). Spike firing frequency was decreased in both sympathetic and parasympathetic MPG neurons, while increased in DRG neurons from the STZ group. However, insulin significantly attenuated the effects of DM on the excitability of MPG and DRG neurons. DM did not alter the passive properties (input impedence, and resting membrane potentials) in MPG and DRG neurons. However, DM significantly increased the duration of afterhyperpolarization (AHP) in MPG neurons, while decreased it in DRG neurons, which might alter the excitability of neurons. Real-time RT-PCR and western blot analyses revealed that expression of T-type 1H Ca2+ channels was down-regulated in MPG, but up-regulated in DRG from the STZ group. Consistent with these molecular data, T-type Ca2+ currents were decreased in sympathetic MPG neurons, but increased in DRG neurons from the STZ group. Furthermore, expression of SK channels determining AHP duration was up-regulated in MPG neurons, while down-regulated in DRG neurons from the STZ group. In in vitro studies, T-type current density was significantly decreased by high glucose and the pro-inflammatory cytokines in MPG neurons. In DRG neurons, however, T-type current density was increased only by high glucose. In addition, H2O2 significantly reduced T-type current density in MPG neurons, while slightly increased it with no statistical significance in DRG neurons. Taken together, experimental DM alters the excitability of autonomic MPG and DRG neurons by differential regulation of expression of T-type Ca2 and SK potassium channels, which might produce autonomic imbalance contributing to the genitourinary dysfunctions. The significances of the present study are as follow; I studied for the first time the effects of DM on functional plasticity of autonomic ganglion neurons innervating the urogenital system. More importantly, I suggest the molecular and cellular mechanisms underlying the DM-induced autonomic plasticity.