The overall goal of the following experiment is to provide practitioners who treat women with pelvic pain, an easy method to highlight the presence and extent of subtle endometriosis. This is achieved by first doing a survey of visible endometriosis to identify obvious disease. As a second step methylene blue dye is applied, which shows areas of subtle endometriosis that might be missed by normal laparoscopy.
Next, all blue stained areas are resected to completely rid the patient of disease. Results are obtained from scanning electron microscopy that show that this approach removes invisible endometriosis that affects the underlying peritoneal cells, which in turn provides menstrual or ovulatory blood access to the sensory nerves that innervate the pelvis causing pain. The implications of this technique extend toward both the diagnosis and treatment of endometriosis as it allows us to identify subtle and even invisible endometriosis that might disrupt the peritoneal surface and allow blood to have contact to the nerves underlying the tissues, explaining the pain that these patients are are experiencing.
Normally at ovulation or menstruation, some blood will enter the pelvic cavity. The lining of the pelvis and abdomen contains an intact sheet of mesothelium that prevents this blood or the factors within blood that cause pain to interact with sensory nerve fibers. In women with endometriosis, the inflammatory endometrioid cells can cause the peritoneal cells to lose their cell cell contacts, providing blood access to the underlying tissues and nerve fibers.
These gaps in mesothelial cells can be seen by scanning electron microscopy, but also are the reason that the blue dye can stain the underlying tissues where it does. I.Women experiencing chronic pelvic pain are first interviewed to ascertain their medical history and details pertaining to the localization of pain timing with regards to the menstrual cycle, associated symptoms including irritable bowel complaints or bladder complaints are noted and recorded. In addition, a physical exam is performed on the patient once patient selection has taken place.
Be sure to ask women undergoing laparoscopy for consent for possible resection of endometriosis and use of their tissues for research. Perform laparoscopy using standard techniques through a five millimeter umbilical port and additional five millimeter ports in the lower abdomen. Carefully inspect the pelvis to correlate any findings of endometriosis with the preoperative localization of pain as indicated by the patient.
Next mixed methylene blue dye one to 200 with sterile saline and use an aspiration needle to inject the solution onto pelvic surfaces. Then perform suction irrigation to remove the excess dye and rinse the peritoneal surfaces. Again, carefully inspect the pelvis and record any areas of blue dye uptake.
When possible, perform a resection of the affected areas of peritoneum. Once the resection is complete, inspect the pelvis to confirm that there is no bleeding. Then release the CO2 gas, remove the instruments and close the incisions with a tissue adhesive.
After resting for an hour in the recovery room, the patient is allowed to go home to document invisible endometriosis by scanning electron microscopy, first suture peritoneal sections to a sterile telfa pad to flatten the specimen and maintain the intraperitoneal side up orientation. Then place the sections in glutaraldehyde fixative for SEM. Once suitably fixed, the samples can be processed for scanning electron microscopy in a different patient with prior resection of endometriosis from the bladder who no longer has pain in that region.
The dye did not stain the peritoneal surfaces, which supports the connection between the correlation of blue dye staining and the presence of pelvic pain. This image of the peritoneal surface was taken from a woman with chronic pelvic pain and bladder complaints that worsened at the time of menses. The laparoscopic image was taken before the application of blue dye and just the reflection of laparoscopic light showed areas suspicious for clear lesions of endometriosis as indicated by the arrows in this image taken in the same area, it can be seen that blue dye applied to the bladder stained distinct portions of the peritoneum.
This image shows a resection of the peritoneum overlying the bladder, and this image shows the cells from this area as seen by scanning electron microscopy. The sample was prepared according to standard procedures and sputter coated with gold platinum alloy to a thickness of 20 nanometers. It can be seen that the peritoneum is coated by cells thought to be endometriosis, and the mesothelial cells of the peritoneum have gaps in their cell cell contacts exposing underlying extracellular matrix.
Whereas this scanning electron microscope image from a normal peritoneum sample prepared in the same manner shows that the epithelial cells of the normal peritoneum are tightly Connected. As you can see, this is a fairly simple technique that you can use with your laparoscopic procedures to help identify subtle and invisible endometriosis that's causing permeability to the peritoneal surfaces. By resecting the entire lesion along with all of the endometrioid cells, you give your patient the best chance for pain relief.
