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From the * Department of Urology, Affiliated Drum
Tower Hospital, Nanjing University, School of Medicine, Nanjing, Jiangsu,
China; and the Department of Urology,
University of Texas Medical School at Houston and MD Anderson Cancer Center,
Houston, Texas.
| Correspondence to: Dr Yutian Dai, Department of Urology, Affiliated Drum Tower Hospital, Nanjing University, School of Medicine, Nanjing 210008, China (e-mail: ytdai{at}hotmail.com) or Dr Run Wang, Division of Urology, University of Texas Medical School at Houston, 6431 Fannin, MSB 6.018, Houston, TX 77030 (e-mail: Run.Wang{at}uth.tmc.edu). |
| Received for publication June 2, 2006; accepted for publication October 30, 2006. |
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Abstract |
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Key words: Diabetes mellitus, erectile dysfunction, intercavernous pressure
Promoting nerve regeneration and preventing nerve degeneration may reverse the neuropathy of diabetic ED. Neurotrophins including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), NT-3, NT-4/5 (NT-4), etc, belong to a protein family with similar structure and function. They are proven to play an important role in the process of repair and regeneration of injured nerves. They also regulate the development and function of postganglionic sympathetic and sensory neurons (Huang and Reichardt, 2001). Te et al (1994) were the first to find NGF in rat deskinned penis. NGF, BDNF, and NT-3 were studied in the major pelvic ganglia (Lin et al, 2003) and in the animal model with pelvic splanchnic nerve lesions (Bakircioglu et al, 2001). Recently, herpes simplex virus (HSV) vector–mediated NT-3 was used to treat diabetic rats, and the ED of diabetic rats was improved (Bennett et al, 2005).
But how do the neurotrophins distribute in the cavernous tissue of control rats, and how do they change in the diabetic rats? Revealing the relationship between neurotrophins and diabetic ED is important to study the pathogenesis of diabetic ED. Therefore, the purpose of this study was to answer those questions.
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Materials and Methods |
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Streptozotocin–Induced Diabetes
Twenty-five male Sprague-Dawley rats weighing 200–250 g were fasted
for 18 hours, lightly anesthetized (ketamine 40 mg/kg IM), and injected
intraperitoneally with freshly prepared streptozotocin (STZ) (Sigma Chemical
Co, St Louis, Mo) (65 mg/kg, n = 15) or vehicle (0.1 mol/L citrate-phosphate
buffer, pH 4.5, n = 10) according to the references
(Dai et al, 2005;
Maeda et al, 1996). Blood
glucose levels were monitored 72 hours later after STZ or vehicle injection,
at regular intervals throughout the study, and immediately prior to sacrifice.
Blood samples were obtained by tail prick, and blood glucose concentration
measured using a blood glucose meter (Roche, Basel, Switzerland). Only those
STZ-induced diabetic rats with serum glucose levels (
16.7 mol/L) were
included in the diabetic group (n = 11).
ICP Measurement
The ICP was determined as described previously
(Ahn et al, 2005;
Shen et al, 2005). Eight weeks
after the injection of STZ, under urethane anesthesia (0.9 mg/kg), the major
pelvic ganglion, cavernous nerves, and pelvic organs were exposed. The skin
overlying the penis was removed, and the right penile crus was exposed by
removing part of the overlying ischiocavernous muscle. A 23-gauge needle
connected to a PE-50 tube with heparinized saline (250 IU/ml) was carefully
inserted into the crus. The other end of the PE-50 tube was connected to a
pressure monitor (RM6042B multichannel signal collection processing system,
Chengdu Implement Company, China). The cavernous nerve was exposed as
described, and electrostimulation (12 Hz, pulse width 5 ms, 5 V, duration 50
seconds) of the cavernous nerve was applied with a stainless steel bipolar
hook electrode. Changes in ICP were measured and recorded by computer.
Sample Collection and Treatment
The penises of the control group and diabetic group rats were removed
without skin and glans by dissection and divided into 2 portions. One segment
was for immunohistochemistry and was immediately fixed into 4% neutral
formalin overnight at 4°C. The other segment was frozen in liquid nitrogen
for later Western blot analysis. The cavernous tissues were then homogenated
with cytosolic fractions and centrifuged. The supernatant was extracted and
mixed with loading buffer. The solution was boiled for 5 minutes and stored at
4°C.
Immunohistochemistry
The samples were processed according to standardized protocols in the
routine histopathology laboratory and according to the references
(Hafidi et al, 1999). Briefly,
after formalin fixation, they were cut into 2-mm slices and embedded in
paraffin. Each 5-µm thin slice was mounted on glass slides, deparaffinized
in xylene, and rehydrated by sequential rinses in absolute, 95%, 80%, and 70%
ethanol. Endogenous peroxidase activity was exhausted by incubation with 1%
H2O2. Sections were then washed in PBS, preincubated in
1% bovine serum albumin for 1 hour, and then incubated overnight on the shaker
at room temperature with a polyclonal primary antibody (1/400 dilution)
directed against 1 of the 2 neurotrophins NGF and BDNF. The sections were then
washed and incubated in biotinylated anti-rabbit or anti-goat secondary
antibody (1/1500 dilution) for 2.5 hours. The signal was amplified with an
acidin-biotin-horseradish peroxidase procedure (vector) and visualized with
diaminobenzidine as the chromogen. Negative control slides were included in
all experiments in which test antibody was omitted and replaced by control
irrelevant diluent.
The immunostaining of NGF and BDNF were performed by gray scale test with a computer assisted image analysis system (HMIAS-2000, Champion Medical Imaging Co, Wuhan, China).
Western Blot Analysis
The samples were processed according to standardized protocols in the
routine histopathology laboratory and according to the references
(Ghinelli et al, 2003). The
total protein solution was separated by 12% or 15% SDS-PAGE and
electrotransferred to a polyvinylidene-difluoride membrane (Bio-Rad
Laboratories, Hercules, Calif). The membrane was blocked with 5% skimmed milk
for 2 hours at room temperature and incubated overnight at 4°C with
primary NGF, BDNF, NT-3, NT-4 (Santa Cruz Biotechnology, Inc, Santa Cruz,
Calif), or ß-actin (Sigma) antibody (1:1000) in confining liquid. After
the membrane was washed (5 minutes x 6) in PBST, it was incubated in the
appropriate HRP-conjugated secondary antibody (Calbiochem, San Diego, Calif)
(1:4000) in confining liquid for 2 hours at room temperature, and followed by
5 minutes exposure in enhanced chemiluminescence (ECL) solution (Pierce
Biotechnology, Inc, Rockford, Ill), exposed to X-ray films (Eastman Kodak,
Rochester, NY), and analyzed using Kodak Digital Science 1D Image Analysis
software (Eastman Kodak).
Statistical Analysis
Student's t test was used to compare the significant difference
between 2 groups. All statistical analyses were processed through the
Statistical Package for the Social Sciences, version 13.0 for Windows (SPSS
Inc, Chicago, Ill). A P value less than .05 was considered
statistically significant.
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Results |
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ICP Determination
There was no difference between control group and diabetic ED group on the
means of baseline intracavernous pressure (P > .05)
(Figure 1). The values of ICP
after electrostimulation of the diabetic ED group were all significantly
decreased (P < .01) compared to control.
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Neurotrophins Protein Expression in Cavernous Tissue
The bands of 4 neurotrophin proteins are shown in
Figure 3 by Western blot
analysis. In diabetic ED rats, 3 of them (NGF, NT-3, NT-4) were all
up-regulated compared to control rats (P < .01). But the contrary
result was found in BDNF; BDNF protein was significantly down-regulated
(P < .01) (Figure 3A through
E).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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In earlier andrology research, neurotrophins were focused on the recuperative factors of injured cavernous nerves. Bakircioglu et al (2001) used the method of intracavernous injection of adeno-associated virus-BDNF to cure the injured cavernous nerve by freezing. They found that it can facilitate the regeneration of the neuronal nitric oxide synthase (nNOS) containing nerve fibers and improve erectile function. After that, several papers were published about the treatment of injured cavernous nerves by administrating BDNF (Hsieh et al, 2003; Chen et al, 2005).
The pathogenesis of diabetic ED is multifactoral, and the neural factor plays a crucial role. In order to exclude the influence of vascular pathological changes, Podlasek et al (2001) used the BB/WOR rat model and found diffuse neuropathic changes in penis and pelvic ganglia. Hecht et al (2001) found that neurological abnormality in men with diabetic ED was as frequent as in men with neuropathic ED. These findings indicate that neuropathy contributes significantly to the pathogenesis of diabetic ED.
How to protect or recover the cavernous nerves damaged under hyperglycosemia is a key problem for us to resolve. Neurotrophins have been proven to be effective in repairing the impairment by surgery or freezing. Since diabetic ED may have similar cavernous nerves injured as neuropathic ED, neurotrophins were expected to have similar changes or effective treatments in cavernous tissue of diabetic ED.
We studied the expression of neurotrophins proteins in cavernous tissue both in control rats and in diabetic ED rats. We found that expression of NGF, NT-3, and NT-4 proteins in cavernous tissue of diabetic ED rats was up-regulated compared to normal control rats, while BDNF was down-regulated. How did these changes happen?
The mechanism of the increase of NGF, NT-3, and NT-4 and the decrease of BDNF is not yet clear. It is one of our research plans in the future. The increase of neurotrophins seems to reflect the degree of cavernous tissue denervation in diabetic neuropathy, and it may represent a compensatory mechanism. We surmise that the increased level of neurotrophins could not completely compensate the severe neuropathy. Consequently the exogenous NGF (Dai et al, 2005) and NT-3 (Bennett et al, 2005) can partly revise erectile function.
The degraded transportation ability of cavernous nerve fibers may be another mechanism of these changes. Neurotrophins were produced by the target tissues, including Schwann cells and endothelial cells, and incorporated with their receptors (TrkA, TrkB, or TrkC) at the nerve ending. Neurotrophin-phosphorylated Trk complex is retrogradely transported to the neuronal body and transduces its signal to the nucleus (Yasuda et al, 2003). However, BDNF is different from other neurotrophins. BDNF is produced not only by target tissue but also by the neuron itself and transported anterogradely (Zhou et al, 1996). Lee et al (2002) demonstrated that streptozotocin-induced diabetes reduces retrograde axonal transport of neurotrophins in the afferent and efferent vagus nerve, so the degraded transportation ability of cavernous nerve fibers may be the important mechanism that caused such a change to the neurotrophins. When the cavernous nerves were injured because of hyperglycosemia, the target tissue produced more neurotrophins (mainly NGF, NT-3, and NT-4) to the cavernous nerve ending, but the increased neurotrophin-phosphorylated Trk complex could not be transported to the upper neuronal body when the cavernous nerves were injured. The BDNF produced by neurons cannot be anterogradely transported to the cavernous nerve ending. Thus the neuron cannot produce and deliver the recover signal to the nerve ending through the axon.
Other interesting results showed that exogenous administration of NGF (Dai et al, 2005) or using HSV vector–mediated NT-3 (Bennett et al, 2005) can partly revise the erectile function of diabetic ED rats. Why were NGF or NT-3 similarly effective as treatments when NGF or NT-3 itself was up-regulated? It is hard to explain this phenomenon at present. We believe that overexpression of neurotrophin can help the formation of neurotrophin-receptor complex. Neurotrophin may also be involved in other similar pathways. Emanueli et al (2003) found that NGF was also a stimulator of angiogenesis and arteriogenesis. The NGF or NT-3 treatment may improve the vascular pathology of diabetic cavernous tissue. More studies are needed to answer these questions.
Is there any relationship between the neurotrophin signaling pathway and nNOS/NO pathway? This problem should be answered in future research work.
As a conclusion, 4 neurotrophins (NGF, BDNF, NT-3, and NT-4) were expressed in cavernous tissues. Three of them (NGF, NT-3, and NT-4) were up-regulated in cavernous tissue of diabetic ED rats compared to control, while BDNF was down-regulated.
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Acknowledgments |
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References |
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|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Bakircioglu ME, Lin CS, Fan P, Sievert KD, Kan YW, Lue TF. The effect of adeno-associated virus mediated brain derived neurotrophic factor in an animal model of neurogenic impotence. J Urol. 2001; 165: 2103 –2109.[CrossRef][Medline]
Bennett NE, Kim JH, Wolfe DP, Sasaki K, Yoshimura N, Goins WF, Huang S, Nelson JB, de Groat WC, Glorioso JC, Chancellor MB. Improvement in erectile dysfunction after neurotrophic factor gene therapy in diabetic rats. J Urol. 2005;173: 1820 –1824.[CrossRef][Medline]
Burgers JK, Nelson RJ, Quinlan DM, Walsh PC. Nerve growth factor, nerve grafts and amniotic membrane grafts restore erectile function in rats. J Urol. 1991;146: 463 –468.[Medline]
Chen KC, Minor TX, Rahman NU, Ho HC, Nunes L, Lue TF. The additive erectile recovery effect of brain-derived 1neurotrophic factor combined with vascular endothelial growth factor in a rat model of neurogenic impotence. BJU Int. 2005;95: 1077 –1080.[CrossRef][Medline]
Dai YT, Chen Y, Yao LS, Yang R, Sun ZY, Wen DG. Expression of nerve growth factor in cavernous tissue and its effects on the treatment of rats with diabetic erectile dysfunction [in Chinese]. Zhonghua Nan Ke Xue. 2005;11: 748 –751, 754.[Medline]
Emanueli C, Schratzberger P, Kirchmair R, Madeddu P. Paracrine control of vascularization and neurogenesis by neurotrophins. Br J Pharmacol. 2003;140: 614 –619.[CrossRef][Medline]
English AW, Meador W, Carrasco DI. Neurotrophin-4/5 is required for the early growth of regenerating axons in peripheral nerves. Eur J Neurosci. 2005;21: 2624 –2634.[CrossRef][Medline]
Ghinelli E, Johansson J, Rios JD, Chen LL, Zoukhri D, Hodges RR,
Dartt DA. Presence and localization of neurotrophins and neurotrophin
receptors in rat lacrimal gland. Invest Ophthalmol Vis
Sci. 2003;44: 3352
–3357.
Hafidi A. Distribution of BDNF, NT-3 and NT-4 in the developing auditory brainstem. Int J Dev Neurosci. 1999; 17: 285 –294.[CrossRef][Medline]
Hecht MJ, Neundorfer B, Kiesewetter F, Hilz MJ. Neuropathy is a major contributing factor to diabetic erectile dysfunction. Neurol Res. 2001;23: 651 –654.[CrossRef][Medline]
Hiltunen JO, Laurikainen A, Klinge E, Saarma M. Neurotrophin-3 is a target-derived neurotrophic factor for penile erection-inducing neurons. Neuroscience. 2005; 133: 51 –58.[CrossRef][Medline]
Hsieh PS, Bochinski DJ, Lin GT, Nunes L, Lin CS, Lue TF. The effect of vascular endothelial growth factor and brain-derived neurotrophic factor on cavernosal nerve regeneration in a nerve-crush rat model. BJU Int. 2003;92: 470 –475.[CrossRef][Medline]
Huang EJ, Reichardt LF. Neurotrophins: roles in neuronal development and function. Annu Rev Neurosci. 2001; 24: 677 –736.[CrossRef][Medline]
Lee PG, Cai F, Helke CJ. Streptozotocin-induced diabetes reduces retrograde axonal transport in the afferent and efferent vagus nerve. Brain Res. 2002; 941: 127 –136.[CrossRef][Medline]
Lin G, Chen KC, Hsieh PS, Yeh CH, Lue TF, Lin CS. Neurotrophic effects of vascular endothelial growth factor and neurotrophins on cultured major pelvic ganglia. BJU Int. 2003; 92: 631 –635.[CrossRef][Medline]
Maeda K, Fernyhough P, Tomlinson DR. Regenerating sensory neurones
of diabetic rats express reduced levels of mRNA for GAP-43,
-preprotachykinin and nerve growth factor receptors, trkA and
P75NGFR. Mol Brain Res. 1996; 37: 166
–174.[Medline]
Podlasek CA, Zelner DJ, Bervig TR, Gonzalez CM, McKenna KE, McVary KT. Characterization and localization of nitric oxide synthase isoforms in the BB/WOR diabetic rat. J Urol. 2001; 166: 746 –755.[CrossRef][Medline]
Sasaki K, Yoshimura N, Chancellor MB. Implications of diabetes mellitus in urology. Urol Clin N Am. 2003; 30: 1 –12.[CrossRef][Medline]
Shen ZJ, Wang H, Lu YL, Zhou XL, Chen SW, Chen ZD. Gene transfer of vasoactive intestinal polypeptide into the penis improves erectile response in the diabetic rat. BJU Int. 2005; 95: 890 –894.[CrossRef][Medline]
Te AE, Santarosa RP, Koo HP, Buttyan R, Greene LA, Kaplan SA, Olsson CA, Shabsigh R. Neurotrophic factors in the rat penis. J Urol. 1994;152: 2167 –2172.[Medline]
Yasuda H, Terada M, Maeda K, Kogawa S, Sanada M, Haneda M, Kashiwagi A, Kikkawa R. Diabetic neuropathy and nerve regeneration. Prog Neurobiol. 2003; 69: 229 –285.[CrossRef][Medline]
Zhou XF, Rush RA, McLachlan EM. Differential expression of the p75
nerve growth factor receptor in glia and neurons of the dorsal root ganglia
after peripheral nerve transection. J Neurosci. 1996; 16: 2901
–2911.
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