| BACKGROUND After studied on one thousand five hundred and eighty twomen beyond forty years old in Shanghai, Len Jing found that the morbidity rate oferectile dysfunction(ED) was 73.1%. The possibility of men beyond forty years oldsuffering from ED is significant higher through epidemic research. There are onehundred and fifty two million men are disturbed by ED. It is supposed that there willbe three hundred and twenty two million men suffered from ED until 2025 in world.Therefore, ED which is bad to the quality of our life should be considered seriously.In all patients of ED, 35%—75% are diabetes mellitus patients. One hundred and fiftymillion people have been diagnosed by diabetes mellitus. It is predicted that thenumber of diabetes mellitus patients will be up to three hundred million. So thedisease of diabetes mellitus with erectile dysfunction has become a high light point toandrology researcher.1. Experimental animal to chooseExperimental animal is the base of medical research because of the definition ofmoral principles, especially in andrology area. So many andrology researchers design different kinds of animal model which are used for research on ED. To guarantee thesimilarity and consistency between animal models and human beings, which animalis suitable is the first problem we should consider. The primate such as monkeys andgorilla, which is the most similar to human beings, is the best experimental animal forandrology research. In our country, there is no report that the primate is used forandrology research, because of expense, lack of animal and moral principals. Rats arethe most common animal in andrology research because they are cheap and similar tohuman beings in anatomy of cavity of pelvis, blood vessel and nerve in penis,distribution of receptor and sensory reflex to sex reaction. Though, rats were chosento research on ED.2. Anatomy of penis and mechanism of erectile functionWithin the penis are two paired corpora cavernosa and a corpus spongiosum.The corpus spongiosum which surrounds the urethra forms the glans penis distally.The corpora cavernosa separate in the perineum to form the crura, which attachbilaterally to the inferior aspect of the ipsilateral ischiopubic ramus. More distally, thecorpora cavernosa communicate in the pendulous penis. Each corpus is surrounded bya thick fibrous sheath, the tunica albuginea, which encases the spongelike cavernosattissue with multiple interconnected lacunar spaces lined by vascular endothelium. Thetrabeculae form the walls of the lacunae and are composed of thick bundles of smoothmuscle and a fibroelasic framed consisting of fibroblasts, collagen, and elastin.Erection follows the relaxation of penile smooth muscle. Dilation of thecavernosal and helicine arteries increases blood flow to the lacunar spaces. Relaxationof the trabecutar smooth muscle dilates the lacunar spaces, causing engorgement ofthe penis. The systemic blood pressure, now transmitted through the dilated helicinearteries, expands the relaxed trabecular walls against the tunica albuginea. Thiscompresses the plexus of subtunical venules, reduces venous outflow in the lacunar space, and elevates lacunar space pressure, making the penis rigid. The pressure in thelacunar space during an erection is the result of the equilibrium between the perfusionpressure in the cavernosal artery and the resistance to blood outflow through thecompressed subtunical venules. The reduction of venous outflow by the mechanicalcompression of subtunical venules is known as the corporal venoocclusivemechanism.Detumescence is the result of the contraction of penile smooth muscle. Theactivation of sympathetic constrictor nerves causes an increase in the smooth muscletone of the helicine arteries and the trabeculae. This results in a reduction of arterialinflow and collapse of the lacunar spaces, with decompression of subtunical venulesand increased venous outflow from the lacunar spaces, returning the penis to theflaccid state.3. Research on phenotype modulation of smooth muscle in corpus cavernosumIn normal men, the content of smooth muscle in corpus cavernosum is 40%—52%. In ED caused by venoocclusive dysfunction, the content of smooth muscle incorpus cavernosum is 19%—36%. In ED caused by shortage of blood supply, thecontent of smooth muscle in corpus cavernosum is 10%—25%. The content ofsmooth muscle in corpus cavernosum will decrease with age.Recently, the research on the role played by smooth muscle on cardiovasculardisease is extensive and intensive. A classic view is that adult vascular smooth musclecells have the capacity to modulate their phenotype, or state of differentiation, in abidirectional manner. This concept was first hypothesized in 1967 by Wissler.Corpus cavernosum is considered as special vascular tissue. Smooth muscle cells intrabeculae have similar function to that in vessels. So whether there is possibility thatsmooth muscle can convert between noncontractile and contractile phenotypes, thereports concerned does not be found. 4. Changes of elastic fiber in tunica albuginea on erectile functionThe tunica albuginea is made up of almost completely of inextensible material; itis able to expand during erection owing to the undulations of its collagenous fibers ina state of flaccidity. The fibers making up the intracavernosal fibrous framework willalso be stretched until the fibers become straight. The sparse elastic fibers may helpthe collagenous fibers of all fibrous elements to regain their undulations in flaccidity.Since the intracavernosal pressure during erection is highly elevated, the arteries andnerves would probably be unable to function if they were not surrounded by fibroussheaths to which the bundles of fibers of the fibrous framework are attached. Thisanatomical arrangement probably prevents the collapsing of arteries and thecompression of nerves during erection, when the elements of the fibrous frameworkstretch. In this latter condition the fibrous bundles of the intracavernsal frameworkpull on the fibrous sheaths around the arteries and the nerves from all directions whilebeing stretched by the distension of the tunica albuginea and its columns during thefilling of the vascular spaces of the corpora when erected. Without the parenchymalframework attached to all fibrous elements of the corpora, and especially to the deepsurface of the tunica albuginea, it is difficult to conceive how the corpora, relativelythin structures compared with their length, would resist flexing and frequent rupturesduring erection and intercourse.5. The contribution of apomorphine (APO) to andrology areaApomorphine is a kind of alkaloid, which is the semisynthesis of morphine andwhose anesthesia effect is lowed down. But its stimulation effect on central nervesystem is stronger, especially on receptor of vomiting in medulla oblongata. So APOis usually to make patients to vomit in clinic. But when rats are injected with APO 30μg per kilogram weight, the erectile function is strengthened obviously by APO. Innormal situation, spontaneous erection is rare in rats. But after injection of micro amount of APO, rats show penis erection, strength, and yawning syndromes. Thesesyndromes usually appear at about ten minutes after APO injection, and can lasttwenty to thirty minutes. Therefore, to inject APO to rats is an effective and good wayto judge rats whether are suffering from ED.Objective 1.how to build diabetes mellitus rats model with erectile dysfunction:①To investigate the best dose of apomorphine(APO) and observation time oferection induced by APO.②To supply erectile dysfunction rats model and (or)diabetes mellitus rats model with standards through research on penile erectionsinduced by apomorphine(APO) and fasting serum glucose.③To construct idealmodel of diabetes mellitus erectile dysfunction(ED) in rats throughstreptozotocin(STZ) injection. 2. To research on morphology of penis tissue in rats ofdifferent moon's age. 3. To investigate phenotype modulation of smooth muscle ofcorpus cavernosum in diabetes mellitus rats with erectile dysfunction. 4. Toinvestigate the changes of elastic fiber in penis tunica albuginea in diabetes mellitusrats on erectile function. 5. To study on the effect of diabetes mellitus onspermatogenesis in rats.Methods1. To investigate the best dose of apomorphine(APO) and observation time oferection induced by APO: Fifteen male SD rats ,each rat was tested with each dose ofAPO in sequence(0μg/kg, 60μg/kg, 80μg/kg, 100μg/kg, 120μg/kg) at sameperiod(9 am to 11 am),one dose per day.2. To supply erectile dysfunction rats model and (or) diabetes mellitus rats modelwith standards through research on penile erections induced by apomorphine(APO)and fasting serum glucose: Thirty SD rats were observed erectile frequency after 100μg/kg APO injection and glucose was recorded after 12 hours fasting. 3. To construct ideal model of diabetes mellitus erectile dysfunction(ED) in ratsthrough streptozotocin(STZ) injection: Thirty male SD rates were randomly dividedinto five groups(control group,STZ 40 mg/kg group,STZ 60 mg/kg group,STZ 80mg/kg group,STZ 100 mg/kg group),6 rats each group. And all rats were observedfour days, one week, two weeks and three weeks after STZ injection. Fasting bloodglucose, erectile frequency induced by apomorphine and change of mass should berecorded.4. To research on morphology of penis tissue in rats of different moon's age:There were four groups, each group had 5 rats. These groups were 1-month-old group,2-month-group group, 3-month-old group and 4-month-old group. All rats' peniseswere cut off at the root of penis after anesthesia. Penises were embedded in paraffin.All slices were staininged by hematoxylin and eosin staining. After the pictures ofslices were collected through digital camera, morphology of penis tissue should bedescribed.5. To investigate phenotype modulation of smooth muscle of corpus cavernosumin diabetes mellitus rats with erectile dysfunction: Diabetes metlitus rats model andwith erectile dysfunction were made through streptozotocin (STZ) injection. 40 ratswere first divided into two groups randomly, 20 rats each that were 4 week group and7 week group. And then divided into four groups, that were control group(5 rats each,without STZ injection), diabetes mellitus rats with erectile dysfunctiongroup(DM&ED group), diabetes mellitus rats without erectile dysfunctiongroup(DM group) and rats without diabetes mellitus and erectile dysfunction groupafter STZ injection(None group). Immunohistochemical staining method and colorimage analysis were used to observe expression of a-actin and desmin in rats corpuscavernosum of different group on the basis of theory that the content of a-actin is akind of sensitive sign of phenotype modulation of smooth muscle in vascular tissue and its decrease accompanies with phenotype modulation of smooth muscle and thecontent of desmin is indirect sign of quantity of smooth muscle cells. Andnoncontractil phenotype is characterized by osteopontin (OPN). So content of OPNmRNA should be detected in situ hybridization.6. To investigate the changes of elastic fiber in penis tunica albuginea in diabetesmellitus rats on erectile function: Diabetes mellitus rats model and with erectiledysfunction were made through streptozotocin (STZ) injection. 40 rats were firstdivided into two groups randomly, 20 rats each that were 4 weeks group and 7 weeksgroup. And then divided into four groups, that were control group(5 rats each, withoutSTZ injection), diabetes mellitus rats with erectile dysfunction group(DM&EDgroup), diabetes mellitus rats without erectile dysfunction group(DM group) andrats without diabetes mellitus and erectile dysfunction group after STZinjection(None group). Victoria blue/Ponceau red staining and color image analysiswere used to observe the content of elastic fiber in tunica albuginea.7. To study on the effect of diabetes mellitus on spermatogenesis in rats: FortySPF rats were used for building diabetes mellitus model induced by treptozotocin(STZ). 40 rats were first divided into two groups randomly, 20 rats each that were 4week group and 7 week group. And then divided into four groups, that were controlgroup(5 rats each, without STZ injection), diabetes mellitus rats with erectiledysfunction group(DM&ED group), diabetes mellitus rats without erectiledysfunction group(DM group) and rats without diabetes mellitus and erectiledysfunction group after STZ injection(None group). Testes were got after anesthesia.And then testes were fixed in 4% paraformaldehyde. Embedded in paraffin, andsections were 5μm. All slices were staininged by hematoxylin and eosin staining.After the pictures of slices were collected through digital camera, morphology ofpenis tissue should be described. Results1. To investigate the best dose of apomorphine(APO) and observation time oferection induced by APO: Significant difference exists in erectile frequency amongfirst 30 minutes group via followed 30 minutes group (F=192.651, P＜0.001), 60μg/kg group via 100μg/kg group (P＜0.05), 60μg/kg group via 120μg/kg group (P＜0.05). There exists no significant difference in erectile frequency among 60μg/kggroup via 80μg/kg(P=0.336) and 100μg/kg group via 120μg/kg group(P=0.813).2. To supply erectile dysfunction rats model and (or)-diabetes mellitus rats modelwith standards through research on penile erections induced by apomorphine(APO)and fasting serum glucose: 95% reference range fasting serum glucose is 4.0 mmol/Lto 7.2 mmol/L.And all rats injected by APO showed penis erection in fist thirtyminutes.3. To construct ideal model of diabetes mellitus erectile dysfunction(ED) in ratsthrough streptozotocin(STZ) injection: Significant difference exists in fasting foodglucose among groups of different test time(F=7.297, P=0.002). There is significantdifference in fasting blood glucose and change of mass between groups of differentdose of STZ injection (P＜0.001).There is no significant difference in erectilefrequency induced by apomorphine and change of mass between groups of differenttest time(F=1.236, P=0.306 and F=0.584, P=0.628).4. To research on morphology of penis tissue in rats of different moon's age:There was much difference between 1-month-old group rats and other 3 groups. In1-month-old group, within the penis were one corpora cavernosa (not two pairedcorpora cavernosa) and a corpus spongiosum. The trabecutae formed the walls of thelacunae and were composed of few bundles of smooth muscle and collagen. In otherthree groups, there was no difference in morphology between these three groups.Penis was formed by two paired corpora cavernosa and a corpus spongiosum, but albuginea septum between two paired corpora cavernosa did not exist.5. Expression of a-actin of smooth muscle in corpus cavernosum in DM&EDgroup is the lowest in all four groups (P＜0.05). No significant difference existsbetween 7 week group and 4 week group(F=0.608, P=0.442), and so doesinteraction(F=2.130, P=0.120). There is no significant difference in expression ofdesmin of smooth muscle in corpus cavernosum between four group( F=0.045, P=0.987) and so do that between 7 week group and 4 week group and interaction(F=-0.045, P=0.987; F=0.572, P=0.639). Expression of OPN mRNA ofsmooth muscle in corpus cavemosum in DM&ED group is the most in all four groups(F=51.648, P=0.000). No significant difference exists between 7 week group and 4week group(F=0.984, P=0.330), and so does interaction(F=0.246, P=0.864).6. To investigate the changes of elastic fiber in penis tunica albuginea in diabetesmellitus rats on erectile function: There exists significant difference between differenttest group (F=10.433, P=0.000). The content of elastic fiber in tunica albuginea inDM&ED group is the lowest in all four groups (P＜0.05). No significant differenceexists between 7 weeks group and 4 weeks group(F=0.685, P=0.415),and so doesinteraction(F=0.905, P=0.452).7. To study on the effect of diabetes mellitus on spermatogenesis in rats: Incontrol group and none group, the arrangement of seminiferous epithelium is regular.The alignment is compact. The architecture is lampros. Proliferation ofspermatogonium is normal, which is laid on the lamina of seminiferous tubuleregularly. But in DM and DM&ED group, the arrangement of spermatogenic cell ineach grade was in disorder. And few spermatocyte, spermatid and sperm can befounded.Conclusion1. The best dose of apomorphine (APO) is 100μg/kg and the best observation time of erection induced by APO is the first 30 minutes after APO injection.2. In rats, erectile dysfunction can not be considered if penile erection exists afterAPO injection, and diabetes mellitus can be considered if fasting serum glucoseexceeds 7.2 mmol/L.3. The best dose of STZ injection to construct ideal diabetes metlitus rats modelis 60 mg/kg and the best time to choose ED rats model is about two weeks after STZinjection.4. There exist difference in penis morphology between human and rat. And themorphology of penis in rats develops with age.5. The architecture of smooth muscle to contract in corpus cavernosum is similarwith that in vascular tissue. Phenotype modulation of smooth muscle in corpuscavernosum can be caused by diabetes mellitus disease. Erectile dysfunction can becaused by phenotype modulation of smooth muscle in corpus cavernosum.6. Decrease of elastic fiber in tunica albuginea can result from diabetes mellitusdisease. Elastic fiber in tunica albuginea plays an important role in the course oferection. Erectile dysfunction can be caused by the decrease of elastic fiber in tunicaalbuginea.7. Diabetes mellitus has a bad effect on spermatogenesis, also can destroy thestructure of seminiferous epithelium. |
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