Evaluation of diaphragmatic mobility following intra-abdominal sub-diaphragmatic fixation of a double-layered mesh in rats 1

PURPOSE: To evaluate the tissue integration of a double-sided mesh after fixation in diaphragm and to study the diaphragmatic mobility by ultrasound. METHODS: Twenty male Wistar rats were used. The animals were assigned into two equal groups according to the day of euthanasia. The animals were anesthetized and a 1.5 x 1.5 cm of double-layer mesh was inserted between the diaphragm and the liver. For the evaluation of the diaphragm mobility a sonographic method was used. Measurements on specific breathing parameters were taking place. Pathological evaluation took place after the animal’s euthanasia. RESULTS: Extra-hepatic granuloma was not differentiated overtime, (χ2=0.04, p>0.05). Neither fibrosis was significantly differentiated, (χ2=0.04, p>0.05). Intra-hepatic granuloma was significantly differentiated overtime, (χ2=10.21, p<0.05). Concerning Te parameter, means were significantly differentiated over time, F (3, 30) = 5.12, (p<0.01). Ttot parameter, it was differentiated over time, F (3, 8)=4.79, (p<0.05). IR parameter was also longitudinally differentiated, F (3, 30)=3.73, (p<0.05). CONCLUSION: The measurements suggest a transient malfunction of diaphragmatic mobility despite the fact that inflammatory reaction, fibrosis and extra-hepatic granuloma were not significantly differentiated with the passage of time.


Introduction
Surgical meshes today represent a group of implants mainly used for hernia repair.In general, the ideal mesh is characterized by a variety of requirements such as economic aspects, functionality and operative handling, sterility or even anti-infective and optimized biocompatibility 1 .
The basic mechanism which explains the broad use is the fibrotic reaction.The ideal mesh should be effective in preventing hernia recurrence; this can be achieved not only by the mechanical action of the mesh, but also by the fibrotic reaction caused by it.
Unfortunately, this fibrotic reaction led to pain and restriction of movement, as well as other clinical complications such as fistula formation and adhesions 2 .This realization led to the concept of a dual-sided mesh to prevent or minimize visceral adhesions on one side while maximizing fibroblast ingrowth and tissue incorporation on the other side.Bilaminar mesh types and composite materials that include a temporary tissue separating layer are now available 3 .
The use of a dual mesh placed intraperitoneally has increased in recent years to treat various hernias forms of the abdominal wall, including hiatal hernia.The intraperitoneal placement is clearly superior than the one on the Myoserosal layer placement due to the lower probability of recurrence (for mechanical reasons), while the problem of creating adhesions and fistulas in the bowel appears to have been addressed through the use of dual grids (with biodegradable internal membrane) 4 .
The purpose of this study is to evaluate mainly the effects of intra-abdominal sub-diaphragmatic fixation of a double layered mesh on the mobility of the diaphragm, by using ultrasonography.
To the best of our knowledge, this effect has not been investigated in the literature up to date.The second aim of this study was to investigate tissue integration of the mesh.

Methods
The study was performed at the Biomedical Research

Prosthetic material
We have used the PROCEED surgical mesh (Ethicon, Somerville, NJ) which is a sterile multi-layered, thin, flexible, laminate mesh comprised of an oxidized regenerated cellulose (ORC) fabric and PROLENE soft mesh, a no absorbable polypropylene mesh which is encapsulated by a Polydioxanone polymer.The polypropylene mesh side of the product allows for tissue ingrowth, while the ORC side provides a bioresorbable layer that physically separates the polypropylene mesh from underlying tissue and organ surfaces during the wound-healing period to minimize tissue attachment to the mesh.The Polydioxanone provides a bond to the ORC layer 5 .

Surgical technique
The animals were anesthetized and surgical anesthesia was maintained throughout the experiment with inhaled Isoflurane (0.5% to 3.0%).The hair was clipped thoroughly immediately before the surgical procedure, followed by antisepsis with Iodopovidone scrub.A 7 cm midline incision was made, caudal to the xyphoid and the peritoneal cavity was entered.A 1.5 x 1.5 cm of the double-layer mesh was inserted in a mostly intact peritoneum between the diaphragm (the nonabsorbable layer) and the liver (the ORC layer) and fixed in the diaphragm using four interrupted 5-0 Prolene suture (Ethicon Inc., Somerville, NJ) placed in each corner so that it would remain fully stretched and smooth and in constant contact with the diaphragm.The abdominal wall and the skin were sewn with a 3-0 polyglactine (Vicryl, Ethicon Inc., Somerville, NJ) simple continuous pattern.All the animals recovered from surgery uneventfully and were included in the study.A single dose of Cefuroxime (25 mg/Kg I.M) was given to the animals postoperatively.

Evaluation of diaphragmatic mobility using ultrasonography
For the sonographic evaluation of the diaphragm's mobility, a standardized method was used [6][7] .We used the GE Vivid I ultrasound (GE Medical Systems Israel Ltd, Tirat Carmel, Israel) with a microconvex probe (8C RS, GE Yokogawa Medical Systems, Ltd, Tokyo, Japan), of a variable frequency of 5 to 13 MHz.In order to achieve a more detailed imaging we used a frequency of 12 MHz, with one focal zone set at a depth of 1.25 -1.75 cm.Two-dimensional mode was used to find the best approach and to select the exploration line of diaphragm.In the

Evaluation of diaphragmatic mobility following intra-abdominal sub-diaphragmatic fixation of a double-layered mesh in rats
Acta Cirúrgica Brasileira -Vol.31 (4) 2016 -237 transverse plane, images were obtained from the midline, just inferior to the xiphoid process, and perpendicular to the abdomen.
The liver was used as an acoustic window.All examinations were recorded on a personal computer for subsequent blind analysis.
The probe was placed in the xyphoid appendix of the anesthetized animal in the subcostal area and was directed medially, cranial and dorsally so that the ultrasound beam reached the vault of the diaphragm that wraps the liver mass.Thus, the inspiratory and expiratory cranial-caudal displacement of the diaphragm respectively shortened and lengthened the probe-diaphragm range.The results concerning the diaphragmatic mobility were analyzed with semi-computerized techniques using the obtained sonographic measurements and the parameters were analyzed as follows (Figure 1).

Study groups and observation periods
The animals (n=20) were assigned into two equal groups according to the predefined day of euthanasia: Ten were sacrificed on 28 th postoperative day (group 1) and ten were sacrificed on 90 th postoperative day (group 2).Sonographic measurements were performed to all animals immediately before the operation took place (1st).Postoperatively, the animal's diaphragmatic mobility was reassessed by sonographic evaluation at 4th (group 1 and 2) 8th and 12th weeks (group 2).At the 4th week (group 1) and at the 12th week (group 2) the animals again were anesthetized and euthanized.A U shape laparotomy was performed and the whole peritoneal cavity was exposed.Liver and diaphragm including the implanted mesh were removed en bloc and fixated for microscopic evaluation.No dissection of the site of implantation was performed to avoid alteration of subsequent microscopic findings.However, no adhesion or seromas nor hematomas were found in the rest of the abdomen.

Histological examination
Five sections of 0.5 cm of every sample were taken including all the structures and tissues and soaked in 10% formalin and then in paraffin blocks.Fine sections (5 μm) were stained

Statistical analysis
Statistical analysis was performed using the Statistical Package for Social Sciences (SPSS 14.0).For the statistical analysis of the diaphragms mobility the means of the repeated assessments of the respiratory curve were compared through a series of subject design models.The Mauchly sphericity test was found to be statistically significant, the differences when assessed through the multivariate test while the aforementioned test was insignificant, the univariate test was used.The effect size was estimated by the η2 coefficient.Chi square test was used to compare percentages.For the histological measurements χ2 was used.

Respiratory curve parameter longitudinal means comparisons: 1st, 4th, 8th and 12th week.
As shown in Table 1 only four respiratory curve parameters were significantly differentiated over time.

Discussion
The use of mesh is not uncommon for the surgical treatment of diaphragmatic hernia.Therefore, it would be of practical interest to know what the changes are regarding diaphragmatic mobility following mesh implantation, given its mechanical actions on the diaphragm and the action of inflammatory changes induced by the mesh.
Proceed Dual-mesh is a lightweight monofilament Polypropylene mesh with large pore sizes.Additionally, The ORC side provides a layer that separates the mesh from the underlying organs and that characteristic minimizes tissue attachment to the mesh.In this respect, although not used in same experimental studies, it was considered a very good choice for our protocol.All meshes produce adhesions when placed adjacent to the bowel, but their extent is determined by their pore size, filament structure, surface area, and even by the patients' individual inflammatory reaction.The pathophysiological mechanism of adhesion formation in the presence of biomaterial in a simplified scheme result from fibrin exudate that follows trauma.The fibrin clots form temporary adhesions that last until the fibrinolytic system absorbs the fibrin with the help of a plasminogen activator.
In our experimental study a lightweight large pore sized mesh was used to prevent contact with the neighboring granuloma leading to a bridging scar.Through statistical analysis, it was found that either inflammatory reaction, neither fibrosis nor extra-hepatic

Foundation
of the Academy of Athens (BRFAA).The experimental protocol was approved by the Veterinary Service of the Athens Prefecture according to the Presidential Decree 160/91-2010 covering the ethical experimentation on animals.Twenty male Wistar rats (Rattus Norvegicus Albinus) were used, weighing an average of 275 gr.(250-300 gr).All animals kept at constant temperature conditions with controlled light/dark cycles, and handled according to the rules established in the BRFAA.
Consequently, the bright line formed by echoes originating from the diaphragm successively moved upwards and downwards on the M-mode graph.The M-mode sonogram was displayed on the video screen with a horizontal sweep speed of 50 mm/sec and was continuously recorded.The measurements in each animal were performed by the same experienced investigator.Both preoperative and postoperative measurements were performed under the same anesthesia protocol.Parameters of diaphragmatic function/mobility which were measured are the following: Parameters of respiratory function o DIA (Diaphragm inspiratory amplitude) o Ti (Diaphragm inspiratory time) o DIV (Diaphragm inspiratory motion velocity) = DIA/Ti o DEA (Diaphragm expiratory amplitude).Same with DIA o Te (Diaphragm expiratory time) o DEV (Diaphragm expiratory motion velocity)=DEA/Te o Ttot (Total breathing time) o DMT (Diaphragm Motion Time).The duration of one breathing curve o DRT (Diaphragm Resting Time) = Ttot-DMT.It represents the calm period of a respiratory cycle o IR (Ti/Ttot) represents the rate of T inspiratory time/ Total breathing time.

FIGURE 1 -
FIGURE 1 -Parameters of diaphragm mobility and respiratory function of the animals.

TABLE 1 -
Respiratory curve parameter means and standard deviations by the 1st, 4th, 8th and 12th week.
Statistical result assessed using the multivariate test as Mauchly W test was statistically significant.*p<0.05,**p<0.01.Means not sharing the same indicator are significantly differentiated according to Bonferroni test, p<0.05.

TABLE 2 -
th week versus 66,7% (2) that belonged to the group sacrificed at the 12 th week.Of the cases that exhibited moderate intra-hepatic granuloma, 14,3% (1) belonged to the group sacrificed at the 4 th week versus 85,7% (6) that belonged to the group sacrificed at the 12 th .No seromas nor hematomas Frequency distribution of intra-hepatic granuloma 4 and 12 weeks after the operation.