Testicular histology after intestinal pedicle flap ( cecum ) apposition in rats

Purpose: Histological study of vascularization between a cecal pedicle flap and the testicle of Wistar rats. Methods: Fifty-three rats were studied. G1: submitted to celiotomy (a), mobilization of the right testicle (RT) to the abdomen (b), cecal flap suture to the RT (d) and cavity closure. G1: procedures a, b and d and fixation of RT into abdomen. G3: procedures a, b and d, exposition of RT to air and reposition into scrotum. G4: not operated. Euthanasia and histology was done after 20 days. Histometry and lesions score classification was done. Testicular vascularization was studied with comparison between G1 and G3. A p < .05 was considered significant. Results: The G1 RT diameters were not different to G2 RT and all have decreased size in comparison with RT of G3 and G4. The lesions score in the RT was 5.83 in G1 and 3.3 in G2 without statistical difference. The vascularization’s average in G1 was 16.9 vessels in 400X field in the RT. In the G3 this average was 0.96 to the RT and 0.92 to left testicles. The weight’s average in G1 was similar with G2 but different of G3 and G4. Conclusion: A significant increase of vascularization was observed between the intestinal flap and the rat testicle.


Introduction
Undescended testis, also called cryptorchidism, is characterized by the absence of the testicle in the scrotum.It is usually due to failure in embryogenesis or in male gonad migration.This condition may also occur due to ischemia or gonadal atrophy1-3 .The incidence is 33% in premature infants, 4% to 5% in live mature births and 0.8% to 1% at the end of the first year of life.Currently, orchidopexy is proposed between 6 months and 12 months of age to bring the gonad down into the scrotum 4,5 .Several efforts have been made to preserve gonad blood flow when performing this kind of procedure, mainly in the cases when it is necessary to ligate the testicular pedicle.One of the possible solutions would be to induce better testicular vascularization, which could increase gonadal blood flow.
The goal of this experimental study is to evaluate, histologically, the testicular vascularization induced by an cecal pedicle flap, sutured to the testicles of Wistar rats and study the testicular lesions resulting from this procedure.We are especially interested in the ideal way (technically) to perform the intestinal apposition to the testicle and induce vascular proliferation.In this experiment we do not seek to prove that increased vascularization found is sufficient to maintain good blood flow to the gonad.

Methods
This experiment was approved by the CEUA (Ethics Commission for the Use of Animals) of the Federal University of Uberlândia (UFU).
Fifty-three adult male Wistar rats (Rattus norvergicus albinus, Rodentia mammalia), aged between 12 and 16 months, apparently healthy, and weighing between 290g and 440g were randomly selected for this study.Seven were excluded after surgical procedures.The animals were supplied by the Experimental Surgery Laboratory of the Operative Techniques, Division of the Medical School, Federal University of Uberlândia (UFU), and were appropriately acclimatized in an experimental environment.They were submitted to a preoperative solid food fasting for 12 hours and weighed thirty minutes before the surgical procedures A Filizola ® balance was used, from 0 to 20Kg and minimal interval of 20g.
Anesthesia was performed with a subcutaneous injection of Cetamine hydrochloride (general anesthetic), using a 0.4 mg /100g dose, in association with a 2% Xylazine hydrochloride solution, with a dose of 0.1mL /100g dose (anesthetic, analgesic and muscle relaxant), according to Laboratory routine and similar to that approved at other experimental laboratories 6,7 .The animals were placed in operative supine position after abdominal hair removal by surgical shaving.
A n t i s e p s i s w a s d o n e w i t h a 2 % a l c o h o l i c polyvinylpyrrolidone iodine solution and a sterile window drape was placed on the shaved area as an aseptic procedure.All the procedures were done following operative techniques of asepsis and antisepsis, including surgical gowns, gloves, masks and caps.The animals were divided into four groups: 12 animals in G1, 12 in G2, and 11 in G3 and 11 in G4, a total of 46 rats.

Pilot study
The original idea was to perform an intestinal flap suture to the gonad, without the mucosal layer, and then replace the testicle back into the scrotum.However, it was impossible to take out the mucosa without provoking flap ischemia.Thus, as we learned in the pilot study, we decided to maintain the mucosa and observe the histological changes resulting from this procedure.A cecal flap was used, and it was impossible to return the testicle to the scrotum, therefore we created a control group (G2) with the testicle placed into the abdomen.

Surgical procedures
Group 1: The rats were submitted to an approximately 3cm-long celiotomy, mobilization of the right testicle to the abdomen by traction.Identification of the cecum, preparation of the cecal flap from a distal area (1 cm) and flap apposition to the right testicle, with a continuous suture of polygalactin-910 (Vicryl ® ) 6-0 (Figure 1).The mucosal surface of the flap was placed in contact with the testicle (albuginea) (Figure 2).Viscera were replaced and the cavity was closed, after testing the suture with a "tire test", with a two-layer suture of polypropylene (Prolene ® ) 4-0.
Group 2: Submitted to celiotomy, mobilization of the right testicle to the abdomen by traction, like in Group 1.The right testicles were fixed in the abdomen (to the peritoneum) with a transfixing suture of polypropylene (Prolene ® ) 6-0, viscera replaced and cavity closed.This group was used as a Group 1 control, where the right testicles were placed into abdomen.
Group 3: Submitted to celiotomy, mobilization of the right testicle to the abdomen by traction.The right testicle was exposed to air for 3 minutes and was replaced in the scrotum.The viscera were replaced and the cavity was closed.
Group 4: The rats were not operated on (control), only separated and observed, as in previous groups and sacrificed for histological evaluation after 20 days.
After surgical procedures the animals were treated with the same chow (Agroceres ® ), ad libitum, and placed in same characteristics environments (cages with food and water).Testicular vascularization was evaluated after the identification of vessels between the cecal flap and right testicle, by microscopy, in five fields of 400X magnification.The Group 1 right testicle vascularization was compared with Group 3 right and left testicle vascularization, considered ideal control.
Tukey tests were used for comparison between averages and Mann-Whitney to evaluate vascularization (Bioestat).The Kruskal-Wallis test was used to compare the lesion scores (Sisvar).A p < .05 was considered statistically significant.The statistical programs used, Biostat and Sisvar, are free license software.

Results
Seven rats were excluded from the study.Four rats from Group 1 and one from Group 3 died and the main cause detected was infection (peritonitis) with intestinal obstruction.One rat was excluded because it was impossible to anesthetize it (G2) and another because of suppuration in the scrotum (right side).Therefore, the testicles of 46 rats (92 testicles) were histologically analyzed.

Testicular diameters
The averages of the largest longitudinal and transverse diameters, in each group were evaluated morphometrically (Table 1).In Group 1, the average longitudinal diameter was 12.75mm in the right testicles (RTLD) and 16.54mm in the left testicles (LTLD).The average transverse diameter in Group 1 was 6.65mm for the right testicles (RTTD) and 9.17mm for the left testicles (LTTD).

Right testicle Left testicle
A statistically significant reduction was observed (Tukey, p < .05) in the diameters of the operated right side testicles from Group 1, compared to the right ones from Group 3 exposed to the air and the right ones from Group 4, which were not operated on.
In Group 2, the longitudinal diameters of the right testicles were 11.41mm.On the left side, the average was 17.31mm.The average testicular transverse diameter in Group 2 was 5.82mm among the right testicles and 9.6mm among the left ones (Figure 3).In the Group 2, the diameters of the right testicles operated on, were also statistically smaller (Tukey, p < .05)than those of the right testicles from Group 3 and right testicles from Group 4 (not operated on).
In Group 3, the average longitudinal diameter of the right testicles (exposed) was 17.4mm and 17.59mm for the same diameter in the left ones.The average transverse diameter of the right testicles was 8.84mm and 9.12mm for the left ones, in Group 3.
In Group 3, the preservation of testicular diameters was observed without a statistical difference (Tukey, p > .05)when compared with the right testicle exposed to air (Group 3) and with those from Group 4 (not exposed).
In Group 4, the average longitudinal diameter of the right testicles was 17.04mm and 16.39mm in the left ones.The average transverse diameter of the right testicles was 9.04mm and 9.11mm, for those on the left.The preservation of the gonadal diameters was also observed, without a statistical difference (Tukey, p>0.05) when we compare right testicles from Group 4 with left ones from Group 4 and with those on the right side from Group 3 (Figure 4).

Testicular lesions
The testicular lesions score was greater in Group 1, where the testicle was placed in the abdominal cavity and sutured to an intestinal flap.In this group, an average of 5.83 points for the right testicles and 0.166 points for the left ones was calculated (only one testicle presented with score = 2).
In Group 2, in which the right testicle remained 20 days in the intra-abdominal position, there were also significant lesions with an average of 3.3 points for the right testicles and zero for the left ones, which were in the scrotum.
In groups 3 and 4 there were no testicular lesions (Figure 5).The statistical analysis in these groups shows that Group 1 does not differ statistically from Group 2 and both differ from Groups 3 and 4 (Kruskal -Wallis, p<0.05).This means that the lesions that occurred in testicles with flaps (cecum) in Group 1 were similar to those that occurred in the testicles placed in the abdominal cavity (Group 2).The operation, which mobilizes the testicle, exposes it to the air and in the end puts it back into the scrotum, did not provoke any testicular lesion (Group 3) (Figure 6).

Discussion
Efforts to preserve testicular blood flow in patients with cryptorchidism, submitted to surgical treatment, have been mentioned in the literature, especially after Fowler-Stephens procedure introduction in 1959.Nowadays, the atrophy rates, resulting from testicular desvacularization, still high [9][10][11] .Autotransplantation was tried, but few results have been showed, especially in small children 12 .
More recently, in the literature, efforts were observed to promote testicular revascularization by using omentum, bladder flaps or fasciovascular flaps sutured to the gonads in experimental models.These procedures have indicated a new way for reducing the rates of postoperative testicular atrophy [13][14][15] .
The experiment results showed us, that it was easily possible to use an intestinal flap with its pedicle, without damage to the gross anatomy of the donor organ.The mobilization of a flap, with a long pedicle, to the scrotum can be very important in high position testicle surgical procedure.Some authors used bladder 14 and omentum [15][16][17] in their experiments.However, we should remember that orchidopexies in human beings have been increasingly indicated for very young boys, about nine months old 18 .At this age, the child's omentum is not highly developed and this condition could result in technical difficulties in descending the testicles into the scrotum.The bladder does not have a vascular pedicle that can be isolated and its wall should occupy the inguinal channel in the case of orchidopexy.In cases of bilateral cryptorchidism, there would not be enough bladder tissue for surgical correction.The instestinal flap could be a good option, to be evaluated in future studies, in which a long vascular pedicle will be necessary to replace the testicles in the scrotum (Fowler-Stephens orchidopexy).
An important observation was that in the testicular lesions variable, there was no significant difference between the right testicles, which were only mobilized and exposed to the air (Group 3) and the right testicles of Group 4, not operated on.According to these observations, handling the gonad or anesthesia and surgical procedures did not contribute to testicular lesions.
Another important fact detected in the experimental model, that placing the right testicles in the abdominal cavity produced the effect of gonadal atrophy with volume reduction in the right testicles of groups 1 and 2, a fact already mentioned in the literature 19 .The suture of the intestinal flap directly on the right testicle in Group 1 and the suture of the right testicle to the peritoneum in Group 2, associated with a rise in the temperature of the testicles positioned in the abdomen contributed to increase the lesion score of these two groups.The lesions in these groups have been larger than in other groups.These data are corroborated by authors who found a significant difference in testicular lesions when direct suturing was applied to the testicle compared with indirect fixation techniques 20,21 .
Good adhesion of the intestinal flap to the testicle was observed in Group 1, and a statistically significant (p <.05) increased testicular vascularization, when we compared Group 1 (flap) and Group 3 (testicular mobilization).
Increased vascularization can be a step to preserve testicular blood flow.However, the findings in this experiment do not demonstrate that intestinal flaps could maintain an adequate testicular blood flow.This hypothesis should be tested in future research, on which Fowler-Stephens procedure must be done.
In a future application, in human beings, the presence of the intestinal mucosa should be avoided.This detail could contribute to a reduction in complications due to infection and neoplastic changes, similar to those which occur in bladder augmentations 22 , using complete intestinal wall.
Another reason to avoid the mucosa layer is the unknown long term effects of the mucus in contact with the peritoneum and viscera.
For technical reasons a cecal flap was used in the experiment.However, it is well known that the best intestinal segment to produce intestinal pedicle flaps to replace the urinary tract, in humans, is the ileum, approximately 20cm to 30cm of the ileocecal valve, as already occurs in bladder augmentation or Mitrofannoff-Monti type intestinal conduits [23][24][25] .The fixation of the intestinal flap seems better when sutured around the terminal portion of the testicular vessels.This has already been mentioned in a publication where omentum was used as a neovascularization inductor [15][16][17] .

Conclusion
A significant increase of vascularization was observed between the intestinal flap and the rat testicle.

FIGURE 3 -
FIGURE 3 -Right and left testicles of one of the rats from Group 2 with different diameters LDRT = Longitudinal Diameter of Right Testicle; LDLT = Longitudinal Diameter of Left Testicle.TDRT = Traverse Diameter of Right Testicle; TDLT = Traverse Diameter of Left Testicle.

FIGURE 4 -
FIGURE 4 -Schematic representation of testicular diameters (mm) in the four groups

FIGURE 5 -
FIGURE 5 -Lesions score averages of each group

TABLE 1 -
Average and standard deviations of testicular diameters in the four groups