| Background and ObjectiveCervical cancer is the world’s fourth most common gynecologic malignancy, according to WHO statistics,500,000 new cases per year, of which 85% of cases in developing countries, while China reached 13.15million, accounting for 28.8 percent of the worldwide new cases of cervical cancer, and was getting younger and younger. About 20 million women die from the disease each year in the world now, which 53000 occurs in our country, threatened the life and health of women seriously.Radical hysterectomy (RH) has become the classic surgical treatment of early stage cervical cancer, it demand excision besides the uterine, and the all tissue around the uterine (include broad ligament,cardinal ligment and uterosacral ligament) and 1/3-1/2 vaginal. However, surgical treatment got the noticeable effect also brought a more serious and irreversible complications, especially under the urethra/bladder dysfunction (sensory loss, urinary storage and voiding dysfunction, urinary incontinence, bladder pressure instability), anal/rectal dysfunction (blood stools, constipation/diarrhea, change in bowel habits) and the vulva/vagina dysfunction (low sexual desire, arousal disorder, orgasmic disorder and sexual pain)etc, all is seriously affecting the patient’s quality of life. PAN from the hypogastric nerve (Hypogastic nerve, HN), pelvic splanchnic nerves (Plevic splanchnic nerve, PSN), lower abdominal plexus (inferior hypogastric plexus, IHP) and its branch, branch rectal, vaginal and clitoral branch and other branches of the bladder issue components. Which is sympathetic hypogastric nerves, bladder smooth muscle relaxation, resulting in bladder urine storage expansion, while contraction of the urethral sphincter to stop urinating, contraction of the anal sphincter, motility inhibition straight; the pelvic splanchnic nerve of the parasympathetic, sympathetic opposite effect, since the contraction of smooth muscle of the bladder and urethral sphincter relaxation at the same time lead to urination, enhance straight peristalsis, and promote defecation; abdominal plexus at the confluence of the hypogastric nerve and pelvic splanchnic nerves from, including a sympathetic and parasympathetic plexus and therefore lower abdominal issued support bladder, rectum branch, vaginal and clitoral branch and other branches both hypogastric nerve and pelvic splanchnic nerve function.A number of studies confirm that the above-mentioned complications mainly due to the damage of the pelvic autonomic nerve (PAN),the main mechanism were:(1) Close to the rear wall of the basin hysterectomy uterosacral ligament (ligament, USL) when hypogastric nerve damage (hypogastric nerve, HN); (2) removal of the uterus near the basin wall cardinal ligament(ligament, CL) when the pelvic splanchnic nerve and pelvic plexus damage; (3) removal of the cervix and vagina bladder ligament (vesico-cervical-vaginal ligament, VCVL) beside the deep and vaginal tissue (collectively, beside the vaginal tissue complex, paracolpium complex, PC) when the bladder support pelvic plexus injury, vaginal support. With the prevalence of cervical cancer increasingly younger and survival rates improvement, how to improve the quality of patients life has become the trend of modern cancer treatment. The preservation of autonomic nervous system pelvic autonomic nerve radical hysterectomy (Systematic Nerve-sparing Radical Hysterectomy, SNSRH) which protect the nerves dominate urinary bladder, rectum and anus, vulva vaginal pelvic organs focus Gynecologic Oncology expert research. Because the unclear of the basis of the surgical anatomy, difficulty of identify nerves during surgery, the different surgical standards, the lack of quantitative criteria after nerve-sparing operation thus can not assess the efficacy fully and can not used in clinical practice widely.The concept of identifing and protecting pelvic autonomic nerve during surgery was proposed by the Japanese gynecologist GangLin since the 1920s,and produced by Linlong in 1960s.Until 1980 Sakamoto et al first reported the surgical procedures in English, and to disseminate this surgery. The he core of procedure is to find and retain the nerve in three-dimensional ring around cervical, and in the early days of the main blood vessels and nerve tissue ligament identification relies on touch, experience and anatomical landmarks, considered main vascular department in the soft upper part of the ligament, and the hard part is nervosa, but the method is the lack of anatomy and histology theory. Then there are scholars use liposuction to expose uterine tissue support structures, to find the main ligaments in the pelvic autonomic nerves and pelvic plexus, but the principle of this method is the destruction of the connective tissue in the ligaments to expose the nerve, but it being questioned whether it will lead to the destruction of tumor tissue impacting the diagnosis and prognosis at the same time. To further understand the precise anatomy of the three-dimensional ring around cervix, this research team and other scholars at home and abroad have the use histopathological methods to research each ligament tissue sections samples and underwent immunohistochemical staining, to clear the composition containing blood vessels, nerves (sympathetic and parasympathetic), lymphatic vessels and fibrous tissue, and to conclude the substantially distribution of the nerves and vessels within the three-dimensional ring around cervix. But the focus of the above approach is that of a single ligament research, only a general description of neurovascular in each ligament, failed to descript the three-dimensional ring around cervix as a whole.In the late 1980s to the early 1990s, the development of the computer image processing technology and biotechnology to make the three-dimensional image of biological tissue reproduction a real. Now, the computer image reconstruction techniques have been increasingly widely used in the field of biology. In recent years, the three-dimensional reconstruction technique has been widely used in the medical field. Three-dimensional structure of biological imaging techniques are the following: serial sectioning technique, NMR (Nuclear Magnetic Resonance, NMR) technology, B ultrasound, CT (Computed Tomography) and so on. However, in addition to serial sectioning technique, the resolution of these imaging methods are a few millimeters thicker magnitude. Therefore, the need to a combination of slice section and three-dimensional reconstruction, to construct three-dimensional structure which can identify and clearly show the structure and other small organizations in three-dimensional ring around cervical. Since 2011, our team found the way to reconstruct the mirostructure in the ligaments using serial tissue slice data sets. But there is not yet a complete three-dimensional digital model of neurovascular in three dimensional ring around the cervix and guide SNSRH surgery.On the basis of preliminary studies, this study is to collect the ligaments after RH and labeling, doing the serial section and staining, combined the image recognition, location registration and 3D reconstruction technology, to rebuild a complete neural and vascular in three dimensional ring around the cervix, thus to provide three-dimensional anatomical basis for SNSRH surgery.Materials and Methods1.SpecimenSelect the patient visit to the Nanfang Hospital in August 2014, and pathologically confirmed squamous cell carcinoma whose cervical cancer stage was IB1, accepting abdominal radical hysterectomy, underwent MRI and CT preoperative, and collect their original scan data. Collect specimens after surgery and the lateral edge of the ligament confirmed by pathology without cancer invasion.2. Drawing materialsAfter removal of the uterus, together with its surrounding ligaments exhibition placed flat plate specimens, identify the main ligaments beside bilateral sacral ligament and vaginal tissue. Removed the bilateral of the sacral ligament, uterosacral ligament and vaginal tissue as a whole, together with some whole cervical tissue. To distinguish the direction, with India ink mark cervical lateral side and edge of the upper of the sacral ligament, and the entire outer surface and the upper edge of uterosacral ligament.3. Embedding, producer and dyeingConventional fixed specimens, after dehydration, take advantage of the needle through four slightly longer than the liver tissue specimens (diameter of about 0.5mm) as the positioning rod mark together with specimens embedded in paraffin, serially sectioned after 30min frozen specimens from the cervical side, slice thickness 5 u m, select 10 continuous sheets as a group and to pick five slides which were not off pieces for HE staining, arterial elastic fibers special staining and immunohistochemical staining (specific marker of sympathetic, parasympathetic, and lymphatics).4. Collection imagesCollect all different stained serial sections images. Use digital slice scanning system for scanning, first slide into 2×10 magnification scanned maps to determine the scan range, and then adjust the magnification to 10 x 10 magnification for automatic scaning, all images stored in BMP format, we collected 640 serial sections data sets in all.5. Image analysisAfter separation of the positioning rod only pictures with Photoshop 5.0 software, using Matlab software for image registration, combined with Photoshop software manual registration adjustment minor differences.6. The three-dimensional reconstructionPut the five groups after registration slice datasets image into Photoshop software, application brush tool to draw contours of blood vessels, sympathetic, parasympathetic and lymphatic. Use ACDSee software images transferred to the appropriate size of each group, were introduced into Mimics software, through images automatic positioning, images organizing, interpolated treatment, reconstructed the blood vessels, sympathetic, parasympathetic and lymphatic in three-dimensional ring around cervical. The method of intravenous digital three-dimensional model reconstruction by Mimics software is using blood vessels masking minus arterial masking, using three-dimensional reconstruction of the direct digital three-dimensional model to reconstruct the vein. Finally, a summary of each model, combined with MRI three-dimensional digital model of the patient’s registration on the model constructed by fusing a complete ring around the cervix within a three-dimensional digital three-dimensional model of neural vasculature. using transparency, measurement, and other functions in Mimics software to analysis the model.Results:1. The qualitative staining of neurovascular in three-dimensional ring around cervix(1) Cardinal ligaments and vaginal tissues:Because when obtaining specimens not be separated, so the study as a whole.① HE staining:Contains a lot of fibrous connective tissue, adipose tissue; rich in blood vessels, medium and small arteriovenous mainly, rich in sinus and single thin-walled veins; rich in nerve tissue; scattered lymphatic occasionally lymphoid tissue and lymph nodes.② Special staining:arterial elastic fibers special staining showed bluish elastic fibers were black, collagen fibers were red, muscle fibers were yellow. Cardinal ligaments and vaginal tissues contains more collagen fiber, bent into a spring-like elastic fibers found in arterial endothelial, muscle fiber content is extremely low, were scattered.③ Immunohistochemical staining:the results of TH antibody specific marker of sympathetic,VIP antibody specific marker of parasympathetic, D2-40 antibody specific markers of lymphatic were all positive. Confirmed that the cardinal ligaments and vaginal tissues include autonomic sympathetic and parasympathetic, including sympathetic ganglia and parasympathetic ganglia. Lymphatic vessels were scattered in no apparent regularity.(2) Uterosacral ligament①HE staining:contains large amounts of fibrous connective tissue, adipose tissue, rich in nerve fibers, blood vessels, to medium and small, micro main vein and blood vessels and nerves associated with multi-line, scattered distribution of lymphatic vessels, lymph nodes occasionally.②Special staining:arterial elastic fibers special staining showed bluish black elastic fibers, collagen fibers were red, yellow muscle fibers. Uterosacral ligament collagen fiber content of more spring-like bent elastic fibers found in the arterial endothelium, less muscle fiber content, and the presence of collagen fibers interlaced.③Immunohistochemical staining:the results of TH antibody specific marker of sympathetic, VIP antibody specific marker of parasympathetic, D2-40 antibody specific markers of lymphatic were all positive. Confirmed that the uterosacral ligament include autonomic sympathetic and parasympathetic, including sympathetic ganglia and a small amount of parasympathetic ganglia. Lymphatic vessels were scattered, occasionally lymph nodes.2. The three-dimensional digital model of neurovascular in three-dimensional rings around the cervicalBased on serial sections of three-dimensional ring around the cervix, combined with three-dimensional computer reconstruction techniques, the three-dimensional digital model of neurovascular in three-dimensional rings around the cervical can be successfully reconstructed. By the analysis and measurement of the model, we found that:2.1 The digital three-dimensional model of each ligament, nerve and vasculature around the cervix(1)The length of the cardinal ligaments and vaginal tissues is 54.60mm, the width is 70.80mm, the thickness is 4.20mm. The cardinal ligaments and vaginal tissues rich in blood vessels at all levels; rich autonomic nerves, including the sympathetic and parasympathetic, sympathetic and parasympathetic were significantly more visible sympathetic and parasympathetic ganglia; lymphatic vessels were scattered.(2)The length of uterosacral ligament is 37.1mm, the width is 25.2mm, the thickness is 4.0mm. Uterosacral ligament contains a large number of blood vessels, to medium and small arteriovenous based; rich in sympathetic ganglia and nerve fibers, including a small amount of parasympathetic nerves and blood vessels associated with multi-line; lymphatic vessels were scattered in small, occasionally lymph nodes.(3)The three-dimensional digital model of vascular:whether in the cardinal ligaments and vaginal tissue adjacent or in uterosacral ligaments, blood vessels are the main line in the upper half of each ligament. Through mimics software measurement:the distance between the upper edge of cardinal ligament and vaginal tissues and artery, from near to far was 6.20mm,4.10mm,3.60mm; vein from near to far was 3.20mm, 0.42mm,5.10 mm. In the uterosacral ligament from near to far was was 8.02mm, 5.03mm,9.50mm; vein from near to far was 10.06mm,6.80 mm,1.60 mm respectively.(4)The three-dimensional digital model of nerves:whether in the ardinal ligaments and vaginal tissues or in uterosacral ligaments, nerves are mainly traveling in the lower half of each ligament, significantly more than the parasympathetic and sympathetic.① Sympathetic:Traveling in the lower half of major ligaments. Through mimics software measurement:the distance between the upper edge of cardinal ligament and vaginal tissues and sympathetic, from near to far was 9.60mm,12.40mm,10.10mm; In the uterosacral ligament from near to far was3.40mm,18.90mm,10.80mm respectively.② Parasympathetic:significantly less than sympathetic, mainly traveling in the bottom half of the ligament. Through mimics software measurement:the distance between the upper edge of cardinal ligament and vaginal tissues and parasympathetic, from near to far was 9.90mm,11.90mm,10.30mm; In the uterosacral ligament from near to far was 16.90mm,18.20mm,19.10mm respectively.(5) The three-dimensional digital model of lymphatic:observed uterosacral ligament content more lymphatic than the cardinal ligament and vaginal tissues slightly, in the uterosacral ligament from the pelvic wall side to the cervical lymphatics shows a growing trend.(6) The three-dimensional digital model of three-dimensional ring around the cervix after registration:the blood vessel main traveling in the upper half of ligaments, the nerve main traveling in the lower half of ligaments, lymphatic were scattered. The distance between the vessels and nerves in cardinal ligament and vaginal tissues from near to far was 0.50mm,1.90mm,3.10mm; In the uterosacral ligament from near to far was 3.90mm,5.81 mm,7.20mm respectively.2.2 Overall cervical ligament neurovascular digital three-dimensional modelThrough the multi-angle, all-round, three-dimensional rotation observed of the three-dimensional model from any viewing angle can show nerves, vessels and traveling relationship in each ligament. The use of measuring tools to accurately show the distance between nerves and blood vessels, thus confirming the major blood vessels to go on line in the second half ligaments, nerves line in the lower half of the main ligaments. And as a basis to construct three-dimensional traveling of PAN in cervical ligaments.ConclusionIn this study, we successfully reconstructed the three-dimensional digital model of neurovascular in three-dimensional ring around cervical combining the paraffin tissue sections with computer technology. By the late registration and fusion technology, the model can be truly show the relationship between the ligament in dimensional space, not only can accurately show the distribution of courses sympathetic, parasympathetic, lymphatic and blood vessels in the three-dimensional ring around cervical, and more able to use the software image scaling and rotation, show the three-dimensional relationship between nerve in each ligament, and can provide a different perspective of the morphological features of any orientation, ease of observers from different angles, different planes, different spatial orientation careful observation, provide a basis for SNSRH surgical procedure standardization. |
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