Comments related to the studies

By Shirli Homburg, PhD 

Hi all,

Sometime ago i have looked in PubMed for adhesions (adhesions and surgery) and thought that, it can be a good idea to go over it from time to time and tell you what is new in the field of adhesion research (basic and clinical research) as I´m as a biologist familiar with the terminology and have a personal intrest in ARD and ARD sufferers…

PubMed is an archive of medicine and life sciences journal literature. It provides a search service of over 11 millions citations and always uptodate. New editions of these professional journals with new publications are published usually every month so I will try every month to look what is new and interesting, mind you there can be some monthes with no new publications concerning pelvic adhesions as its still (unfortunately) not one of the “hottest” research subjects, although it seems now, to be more “fashionable” topic than ever… so if there are no new papers about adhesions at that month, I will choose others, as even one year old paper (and even older) can be still “new” and original if no one else added to the specific subject.

Well !, the“paper of the month”: “Effect of melatonin in the prevention of post-operative adhesion formation in a rat uterine horn adhesion model”. The work was published in August (2003) in a prestige journal (Human Reproduction) by the group of Narin, Tayyar and co. The results are very good, meaningful and applicative (even in the short run), but we shouldn’t forget (as you can read in the title) that the experiments were done in rats and not in humans, although in this case, there is a good chance that these results will repeat themselves more or less in the same way in humans. 
First, some background (which can also be relevant for some future papers) Inflammation is considered to be a normal mechanism of the body to heal itself after injury ( an operation in this context is defined also as an injury). 
An inflammation governs many procedures and infact is a multipathway procedure (including blood clotting, fibrinolysis of blood clots and angiogenesis- rebuilding of capillaries and vessels) in which each pathway is a multistep one.This all involves many and different types of cells. Theses cells, inflammatory and immune cells, mesothelial cells and fibroblast, release molecules that modulates the subsequent evolution of the peritoneal lesion to normal healing or to adhesion formation (depends on there amount and timing..) . Some of these molecules which are then released are the ones that are termed free radicals or oxygen –derived free radicals(like superoxide anion and hydrogen peroxide). You have probably already heard about them in the context of causing wrinkles,aging, mutations and therefore cancer, vitamins that fight them (neutrlize them and make them less toxic) and now adhesions. 
Free radicals are highly toxic by-products of normal metabolism in every cell.They are small and very reactive molecule as they are very negatively charged and therefore need to stabilize by a reaction that let them give away their extra electrons. Free radicals usually react with oxygen ( their main electron acceptor) and by that they are converted to oxygen-derived free radicals which are less reactive- destructive molecules although still very harmful,but at this stage (as oxygen-derived free radicals) the body has special enzymatic mechanisms to get rid of them. When the free radicals and the oxygen-derived free radicals are not efficienty removed or metabolized (especially when their amount is too high) they will react with or attack any macromolecules like DNA or fattyacids in the cell membrane and proteins. Consequently a wide variety of cells including endothelial cells (cells that cover blood vessels and soft tissues), erythrocytes (red blood cells), platelets and fibroblasts (participate in blood clotting and fibrinolysis) are damaged and therefore several procedures are getting totally out of balance … 
A surgery ( so called “injury” ), as was mentioned before, will intiate healing mechanisms (inflammation) and as there is no good without bad some of the cells which are involved In the inflammation mechanisms will produce together with there beneficial activity free radicals that will add to their normal level (normal metabolism). These free radicals react rapidly with oxygen to produce the less toxic (although still very harmful) oxygen-derived free radicals, by that exacerbating the oxygen deficitcontributing to an Ischemia situation (lack of oxygen in and around the cells) which is anyway approaching by using CO₂ gas as is usually being done in an endoscopic surgery (!)… Already after 5 minutes of ischemia there is a significant production of free radicals that have not enough oxygen to react with (free radicals cannot reduce their toxicity by reacting with CO₂ nevertheless CO₂ by his presence lowers the concentration and availability of oxygen to the cells). Therefore free radicals will start attacking those cells which were mentioned above and by that initiating adhesion formation, starting with cytolysis of these cells (cells are broken) and peroxidation of lipids in cell membrane that lead to an increase in the vascular permeability that cause among other things (damage that produce adhesions) also animbalance in fibrin deposition and fibrin dissolution (blood clotting and dissolution of the clots) which produce fibrinous adhesions ( “hard” and more problematic adhesions). Until a certain stage the body can handle and repair at least some of the damage of the free radicals and later on adhesions. 
The body can lyse (“soft”) adhesions by endogenous fibrinolytic activity but the longer the imbalance of systems lasts the body loses it ability to handle the accumulating damage and more adhesions will be formed and more adhesions will be harden (fibrinolytic activity goes down) and just to make it worse when there is an impairment in the fibrinolytic process it signals for help and more cells from the immune system and cells which are involved in angiogenesis (rebuilding of blood vessels) arrive and accelerate the adhesion situation by producing more free radicals on one side and supporting existing adhesions by giving them better blood supply.It is kind of vicious circle. 
Sometime after the “injury” is ended the body heals itself but the fibrinous adhesions stay and can even get harder….

What is melatonin ?
Melatonin is an hormone. It is synthesized in the body, in the brain, by the pineal gland. Melatonin is mainly known to have a role in the regulation of circadian rhythmus (sleeping) and for this you can buy melatonin in any drugstore (!) 
Perhaps it has also a role in reproduction, tumour growth and ageing. 
The most intresting discovery for us is that recently it was proved that Melatonin has also a free radical scavenger activity, as it is a anti- oxidant like vitamin E or C and most important, it is the strongest free radical scavenger or anti- oxidant which have been detected to date. Melatonin as an anti-oxidant can “catch” the free radical,react with it and by that stabilize the free radical so it is no longer toxic to the cell. Except of that Melatonin can even stimulate several antioxidative enzymes (that break the oxygen-derived free radicals, so they are not dangerous any more). 
In other words, it is probably the ultimate free redicals fighter. 

what did they want to investigate? 
Like other adhesions researchers they wanted to find a way to prevent as much as possible adhesions formation after surgery. Being more and more aware to the primary role of excessive free radicals in adhesion formation, it made sense to find a way to reduce their amount and by that their damage. 
The researchers knew they could do it in the same way the body is dealing with free radicals. The body can eliminate free radicals (until a certain physiological level) with antioxidants like vitamins and antioxidants enzymes. As the researchers had the new information about the potential of melatonin,they thought that using such astrong anti free radical substance in a high dosage (it exsists in the body in a low concentration) can counter very high amount of free radicals produced during an operation. They chosed to do it in a known model for adhesions- rat uterine horn (a rat has an uterus in a shape of 2 horns). 
Not only they wanted to investigate the effects of Melatonin on the prevention of adhesion formation (in rat uterine horn) ,they also aimed to determine whether different methods of administration of Melatonin were beneficial. 
There were 7 groups in the experiment : 4 treatment groups : 2 groups that got melatonin, one 30 minutes prior to surgery and the other before closing the abdomen. Another 2 groups having the same treatment as was just descrised but with extra treatment added for both- a daily dosage of melatonin for 5 days after the operation. 3 control groups: one that got nothing, another that got a physiological solution (instead of Melatonin) 30 minutes prior to surgery and the other group got also physiological solution before closing the abdomen.

results
The extent, severity and total scores of adhesion were found to be significantly reduced in all of the Melatonin treatments groups when compared with controls. 
There were no statistically significant differences between the treatments groups (although daily administration of Melatonin for 5 days after surgery seemed to be much more effective in the reduction of adhesion, no statistically (and that what counts) significant difference was detected in the adhesion scores in comparisons with the groups treated with single dose of Melatonin). The conclusions : This study showed that Melatonincan act as an endogenous antioxidant and even a single dose Melatonin therapy was effective in the prevention of post-operative adhesion formation. 

Why are the results important ? what next ? and last comments
It seems now that in general one of the most common and unpleasant complication of pelvic surgery are adhesions. One of the main intiators of these adhesions are free radicals. The reaserch here proved the hypothesis that antioxidants and and free radical scavengers like Melatonin are essentially effective in the first step of adhesion formation. It is the first experimental study that was done with Melatonin on post operation adhesion formation and infact almost the first that was done with a substance as antioxidant against adhesion formation(!) (one work used vitamin E as a free radical scavenger in preventing adhesion formation but the results were not clear). Many different substances have been used in the prevention of adhesions but no satisfactorily effective substance has yet been found, therefore the results of this research are so important and intresting. By the way materials like spraygel are something else, their action are mechanical and not biochemical. 
You can buy Melatonin in your next drugstore and until now, no serious side effects or risks have been reported in association with the use of Melatonin, but before you run and buy some, one has to remember that Melatonin`s effect on adhesion is at the stage of adhesion formation means they can help just before or during or just after operation and ofcourse the results should be proven again in human subjects… (good chance they will). In the next studies also other antioxidants should be examined. 
From time to time we think about or we hear a question like why have someone developed more adhesions or more severe ones than the other ? except of known variables like longer or more difficult operation,bigger cut…. the usual answer one gets is that every individual is a little different than the other (or it’s a matter of personal tendency). It sounds quite vague but it is very true and the explanation lays in this text. We all have the same systems, cells and enzymes but in each one of us the efficiency of different activities done by these systems, cells, enzymes are different, and this is how each individual is build, born with, its genetic. For example, an enzyme that metabolize oxygen-derived free radicals and destroy their harmful effect can be more active in some and less in other, this can then have an effect on how much free radicals the system or the enzyme can deal with. If it cant relatively metabolize a lot of free radicals, more damage will be done by them, which maybe together with other “slow” enzymes can lead to adhesion formation (and other things) in a situation that causes high amount of free radicals like during inflammation after injury (as operation).

And just last thing… If an operation is needed ( to remove adhesions or for any other reason) not to forget that the production of the free radicals can be also reduced by using a suitable operation technique like Gasless as it is a minimal invasive technique and avoids a great “injury” like in laparotomy (open-big cut surgery) which results in a high production of free radicals. nevertheless gasless laparoscopy also avoids the CO₂ gas which is used in gas-laparoscopic surgery and highly contributes to free radical production and therefore inducing adhesion fromation. 
Gasless laparoscopy as an endoscopic surgery without carbon dioxide gas, in huge extent, decreases adhesion formation after surgery by decreasing free radicals and so contributing beside Melatonin to the fight against free radicals.

That’s all for now , take care,

yours Shirli 

Hello again,

 
The paper i want to present here was published less than one year ago in one of the top scientific – gynecological magazines, “Fertility and Sterility” by the group of Molinas and Koninckx from Belgium. The title of this paper (which is rather complicated to a non-professional ear) : “ Role of vascular endothelial growth factor and placental growth in basal adhesion formation and in carbon dioxide pneumoperitoneum-enhanced adhesion formation after laparoscopic surgery in transgenic mice”. 
The results of the work presented in this paper are quite impressive and meaningful. Thus, contributing another stone in finding and understanding the causes to adhesion formation as the researchers proved that stimulation of angiogenesis (the formation of new blood vessels extending from exsisting vessels) and more precise the proteins that are termed angiogenic factors are involved in creation of adhesions. The work also supports past finding about hypoxia (lack of oxygen induced free radicals-see last report) and appearance of angiogenic factors by giving an indirect link between these factors and hypoxia and a direct link between elevation of angiogenic factors and carbon-dioxide used in laparoscopic surgery. 
The impressive impact of this work is also influenced by the fact that the researchers used a relatively new and powerful biological research technique – the use of transgenic mice. That is by itself a step jump in adhesion research.

B a c k g r o u n d : As was already mentioned last time, there are few physiological procedures that are involved in different stages of adhesion formation. These procedures are part of healing processes after an injury (= an operation). When the balance for each procedure is much disturbed by a high hypoxia (lack of oxygen that produces very reactive, and dangerous free radicals) caused by a strong inflammation (inflammation will be after every injury), stiffed adhesions will be formed. For example I will describe in short the known scenario that can cause adhesions by members of the blood clotting system. After peritoneal injury, hypoxia generated by inflammation will trigger formation and activation of several proteins that belong to the blood clotting system like (PAI-1, tPA, TGF-ß, MMPs and TIMPs) when the hypoxia is strong or continues for long, the speed of fibrin degradation is shifted and become insufficient, as a result the remaining fibrin will serve as a scaffold for fibroblasts growth, extracellular matrix deposition and capillary growth leading to adhesion formation. The longer the imbalance process exists the stronger effect it induces… 
Angiogenesis is one of the procedures that is very likely to be involved in adhesion formation however, until now its role in creation of adhesions has barely been explored after laparotomy (the old fashioned “big cut-scar” operation) or laparoscopy (the newer small cut-scar operation). What has been shown until now (as it was shown for in the example above) is that hypoxia, elevate the production of angiogenic factors and by that the process is regulated. These angiogenic factors which their expression are then being modulated are members of the Vascular Endothelial Growth Factor family. This family includes the VEGF-A (vascular endothelial growth factor), VEGF-B and PIGF (placental growth factor). 
The effect of these proteins on adhesion formation was examined in this work. 
The models that were chosed to work with, in order to investigate the role of VEGF-A, VEGF-B and PIGF in adhesion formation, were transgenic mice. 
What are they? They are normal mice except for the fact that with special technology one gene in their cells (a gene is translated to a certain protein which is responsible to a certain function) is deleted or designed to be overexpressed. This is an excellent way to examine an effect in the whole animal (in contrast, for example to tissue culture). In this work the researchers managed to create a group of mice that overexpress the VEGF-A gene, a group with a deleted gene for VEGF-B and another group with deleted gene for PIGF. These transgenic mice groups were always compared after treatments to mice which are termed wild-type mice, mice that nothing in their genome was changed they have normal expression of VEGF-A, a gene for PEGF-B and for PIGF. 
Carbon-dioxide which is used in laparoscopic operations is now a known factor that excelerates adhesion formation probably by contributing (much) to the hypoxia which is generated anyway by injury and inflammation. To this hypoxia as explained before all factors written above are very sensitive. The researchers in this work termed adhesions that are formed as a result of the hypoxia which is caused mainly by inflammation (without any other interference)- b a s a l adhesions. Adhesions that were also caused by using carbon oxide during operation were termed, as reflected in other works- e n h a n c e d adhesion formation. The reasons (mechanisms) for enhanced adhesion formation by carbon dioxide can be the same as in basal adhesion formation only being longer sustained or stronger activated or other mechanisms are involved.

The A I M then of the experiments here were to investigate the role of the angiogenic factors VEGF-A, VEGF-B and PIGF in both basal adhesions and carbon dioxide (pneumeperitoneum)- enhanced adhesions by using transgenic mice (some overexpressing VEGF-A some without VEGF-B and PIGF) that passed laparoscopic surgery. The formation of adhesions in the transgenic mice compare to ones in the wild-type mice (which were also operated) had to be then scored and compared. 
Induction of enhanced adhesion formation or basal adhesion formation was dependent on the duration of the laparoscopic operations. For getting enhanced adhesion formation 60 minutes of laparoscopic operation were required and only 10 minutes were required for getting basal adhesion formation in the transgenic and wild type mice.

R E S U L T S and C O N C L U S I O N S : In comparison with the wild type mice, basal adhesions were higher in the transgenic mice group that over expressed VEGF-A, which means VEGF-A has a direct role in basal adhesion formation (just as a result of an injury-surgery- inflammation). Transgenic mice that lacked VEGF-B or PIGF had similar basal adhesion formation as the wild type, indicating they are not involved in basal adhesion formation (but as other results indicate in carbon dioxide enhanced adhesion formation). All wild type mice that had a longer operation with carbon dioxide had enhanced adhesion formation. In all groups of transgenic mice carbon dioxide did not enhanced adhesion formation as expected. It was also acceptable in the overexpressed VEGF-A mice because the total effect of VEGF-A on general adhesion formation is probably near maximum as it was already been activated by the inflammation induced hypoxia at the beginning.

The results of the study confirms previous finding that carbon dioxide is a cofactor in adhesion formation. The data here demonstrate that the VEGF family plays a role in adhesion formation. VEGF-A has a direct role in basal adhesion formation and all have a role in enhanced adhesion formation induced by carbon dioxide, implying that up-regulation of these three angiogenic factors can serve as a mechanism for the enhanced adhesion formation by CO₂ gas used in laparoscopic surgeries.

And L A S T, although these results, that were published for the first time seems now to you a little abstract, one should remember that any information about causes and mechanisms (even in mice) can open ways to produce in the future a treatment or a prophylactic treatment against adhesions, maybe not by producing transgenic humans but for example by generating antibodies that will catch some of the proteins that are involved in adhesion formation….

Questions / comments are welcome. 
Until next time, all the best,

 
Shirli. 

Researchers from the department of surgery in the Children´s hospital Boston led by Dr. mark Puder have just reported and published that painful surgical adhesions may be preventable by Celebrex® a common oral arthritis drug. Its a Cox-2 inhibitor ( cox-2 an enzyme that participates in many inflammatory processes) and taking it just before and immediately after surgery has a clear anti adhesion effect. 
The researchers tested tested COX-2 inhibitors in an animal model of abdominal adhesion formation. After undergoing surgery, groups of 6 to 18 mice received either COX-2 inhibitors (Celebrex or Vioxx), non-selective COX inhibitors (such as ibuprofen and aspirin) or placebo for 10 days. . 
The results were dramatic : At 10 days, the placebo group had obvious abdominal adhesions. Mice receiving non-selective COX inhibitors had a slight reduction in adhesions, and the COX-2 inhibitor group had a larger reduction. The greatest reduction was in the mice given Celebrex, and 6 of 11 Celebrex-treated mice (55%) were completely adhesion-free. The researchers then observed the Celebrex, Vioxx, aspirin and placebo groups for an additional 25 days. Again, the Celebrex group had the fewest adhesions. The adhesion score (a measure of both the extent of adhesions and the difficulty of removing them) was only 1 in the Celebrex group, 5 in the Vioxx group, 8 in the aspirin group, and 11 in the placebo group.

Based on these findings,Dr Mark Puder is preparing to set up a multi-institutional clinical trial of Celebrex in adult surgical patients. If Celebrex works in humans,the doctor could give it to patients on the day of abdominal surgery and the 10 days after surgery,recommended Dr. puder.

Celebrex has all best chances to work in humans : COX-2 inhibitors are anti-inflammatory drugs best known for their use in arthritis. However, they also inhibit angiogenesis, or formation of blood vessels, and Celebrex also inhibits fibroblast activity, important in scar formation. These three properties make Celebrex a particularly good candidate for testing, since adhesions are made up of inflammatory cells, blood vessels, and fibroblasts. By using celebrex then each component of the adhesion is stopped, then within five days the tissue surface can get a whole new lining of mesothelial cells, Once these cells resurface the area, adhesions won't form."

Like in many important discoveries it was all found accidently : Puder and colleagues did not set out to study adhesions. They were studying the effects of Celebrex on liver regeneration after injury to the liver. When they went to examine the livers of the mice, they noticed to their surprise that there were no adhesions, an unusual finding. So they decided to do a formal experiment…..

We are looking forward to collaborate with Dr.Puder and meanwhile we check the possibility to use celebrex here in Germany as an off the label drug for adhesion patients. News will be informed.

 
Yours, Shirli.