Stable Gastric Pentadecapeptide BPC 157 Therapy for Primary Abdominal Compartment Syndrome in Rats

Recently, the stable gastric pentadecapeptide BPC 157 was shown to counteract major vessel occlusion syndromes, i.e., peripheral and/or central occlusion, while activating particular collateral pathways. We induced abdominal compartment syndrome (intra-abdominal pressure in thiopental-anesthetized rats at 25 mmHg (60 min), 30 mmHg (30 min), 40 mmHg (30 min), and 50 mmHg (15 min) and in esketamine-anesthetized rats (25 mmHg for 120 min)) as a model of multiple occlusion syndrome. By improving the function of the venous system with BPC 157, we reversed the chain of harmful events. Rats with intra-abdominal hypertension (grade III, grade IV) received BPC 157 (10 µg or 10 ng/kg sc) or saline (5 ml) after 10 min. BPC 157 administration recovered the azygos vein via the inferior–superior caval vein rescue pathway. Additionally, intracranial (superior sagittal sinus), portal, and caval hypertension and aortal hypotension were reduced, as were the grossly congested stomach and major hemorrhagic lesions, brain swelling, venous and arterial thrombosis, congested inferior caval and superior mesenteric veins, and collapsed azygos vein; thus, the failed collateral pathway was fully recovered. Severe ECG disturbances (i.e., severe bradycardia and ST-elevation until asystole) were also reversed. Microscopically, transmural hyperemia of the gastrointestinal tract, intestinal mucosa villi reduction, crypt reduction with focal denudation of superficial epithelia, and large bowel dilatation were all inhibited. In the liver, BPC 157 reduced congestion and severe sinusoid enlargement. In the lung, a normal presentation was observed, with no alveolar membrane focal thickening and no lung congestion or edema, and severe intra-alveolar hemorrhage was absent. Moreover, severe heart congestion, subendocardial infarction, renal hemorrhage, brain edema, hemorrhage, and neural damage were prevented. In conclusion, BPC 157 cured primary abdominal compartment syndrome.

Considering the effects of BPC 157 therapy peripherally and centrally (Vukojevic et al., 2018;Gojkovic et al., 2020;Kolovrat et al., 2020;Gojkovic et al., 2021a;Knezevic et al., 2021a;Knezevic et al., 2021a;Gojkovic et al., 2021b;Knezevic et al., 2021b;Strbe et al., 2021), in rats with severely increased intra-abdominal pressure, i.e., primary abdominal compartment syndrome, we attempted to introduce a therapy for compressed essential vessel tributaries, both arterial and venous (peripherally and centrally), due to occluded major veins and arteries, in order to prevent the consequent noxious syndrome, both peripherally and centrally. Otherwise, intra-abdominal hypertension adversely affects many organs, such as the brain, heart, lungs, kidneys, and gastrointestinal tract (Cullen et al., 1989), progressing to lethal levels. As abdominal compartment syndrome leads to organ failure at an intra-abdominal pressure of 20 mmHg (Hunter and Damani, 2004;Hedenstierna and Larsson, 2012), to assess the degree of severity that can be treated with this therapy, higher intra-abdominal pressures of 25, 30, 40, and 50 mmHg were also used. It was found that systemic and splanchnic blood flow and afferent hepatic flow were reduced as the intra-abdominal pressure rose; i.e., liver blood flow decreased by 39% when pneumoperitoneum increased from 10 to 15 mmHg and liver ischemic injury occurred (Chen et al., 2017).
Furthermore, as an immediate effect, the abdominal, thoracic, and cranial cavities interact with each other (Depauw et al., 2019), and increased intra-abdominal pressure causes an increase in intracranial pressure (Malbrain and Wilmer, 2007;Scalea et al., 2007;Youssef et al., 2012;Chen et al., 2020). Increased intraabdominal pressure also increases intrathoracic pressure, which is rapidly transmitted up through the venous system, thereby further increasing intracranial pressure (Malbrain and Wilmer, 2007;Scalea et al., 2007;Youssef et al., 2012;Chen et al., 2020). Thus, although not specifically indicated, these findings support the rapid improvement of venous system function as an essential common point to prevent and reverse the noxious chain of events and attenuate all harmful consequences.
The many blood vessels identified as being activated by specific pathways following a given vessel injury require a regularly applicable therapy, with beneficial effects dependent on, but not limited to, occlusion of a particular vessel . With BPC 157 therapy, this point was envisaged by the consistent reduction of the whole "occlusive-like" syndrome that regularly follows the intragastric application of absolute alcohol in rats  and intraperitoneal application of the lithium overdose (Strbe et al., 2021). Consequently, we observed that this beneficial effect, after direct injury (permanent ligation) applied to one or two major vessels, could instantly oppose more general damage (maintained intra-abdominal hypertension, either high (grade III) or very high (grade IV)), as all blood vessels which can be compressed with increased intra-abdominal pressure. Therefore, a "bypassing key," i.e., an activated azygos vein as a rescuing pathway, avoiding both the lung and liver and also noted in Budd-Chiari syndrome (i.e., suprahepatic occlusion of the inferior caval vein) , combines the inferior caval vein and superior caval vein via direct blood delivery. Thus, activated azygos vein shunt could reorganize blood flow and instantly attenuate the consequences of maintained high intra-abdominal pressure, both peripherally and centrally.
Thus, we assessed BPC 157 therapy as a curative principle in rats with established permanent intra-abdominal hypertension. As confirmation, we used the crisis that occurred with the high intra-abdominal pressure-induced syndrome, in which intraabdominal hypertension simultaneously affected all abdominal vessels and organs for a considerable period and restrained the ability to recruit alternative pathways, such that a deadly situation was created before therapy initiation.

Animals
This study was conducted with 12-week-old, 200 g body weight, male Albino Wistar rats, randomly assigned at six rats/group/ interval. Rats were bred in-house at the Animal Pharmacology Facility, School of Medicine, Zagreb, Croatia. The animal facility is registered with the Veterinary Directorate (Reg. No: HR-POK-007). Laboratory rats were acclimated for five days and randomly assigned to their respective treatment groups. Laboratory animals were housed in polycarbonate (PC) cages under conventional laboratory conditions at 20-24°C, relative humidity of 40-70%, and noise level of 60 dB. Each cage was identified with dates, number of studies, group, dose, number, and sex of each animal. Fluorescent lighting provided illumination 12 h per day. Standard good laboratory practice (GLP) diet and fresh water were provided ad libitum. Animal care was in compliance with standard operating procedures (SOPs) of the Animal Pharmacology Facility and the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes (ETS 123).
This study was approved by the local ethics committee. Ethical principles of the study complied with the European Directive 010/ 63/E, the Law on Amendments to the Animal Protection Act (Official Gazette 37/13), the Animal Protection Act (Official Gazette 135/06), the ordinance on the protection of animals used for scientific purposes (Official Gazette 55/13), Federation of European Laboratory Animal Science Associations (FELASA) recommendations, and the recommendations of the Ethics Committee of the School of Medicine, University of Zagreb. The experiments were assessed by observers blinded as to the treatment.
Recordings of brain swelling were performed in rats before sacrifice after complete calvariectomy was performed Knezevic et al., 2021a;Knezevic et al., 2021a;Knezevic et al., 2021b). Briefly, six burr holes were drilled in three horizontal lines, all of them medially to the superior temporal lines and temporalis muscle attachments. The two rostral burr holes were placed just basal from the posterior interocular line, the two basal burr holes were placed just rostral to the lambdoid suture (and transverse sinuses) on both sides, respectively, and the two middle burr holes were placed in line between the basal and rostral burr holes.
Rats were laparatomized before sacrifice for the corresponding presentation of the peripheral vessels (azygos vein, superior mesenteric vein, portal vein, inferior caval vein, and abdominal aorta). The recording was performed with a camera attached to a VMS-004 Discovery Deluxe USB microscope (Veho, United States) at the end of the experiment and assessed as before Knezevic et al., 2021a;Knezevic et al., 2021a;Knezevic et al., 2021b;Strbe et al., 2021).
Superior Sagittal Sinus, Portal, Superior Mesenteric, and Caval Vein, and Abdominal Aorta Pressure Recording As described before (Vukojevic et al., 2018;Gojkovic et al., 2020;Kolovrat et al., 2020;Gojkovic et al., 2021a;Knezevic et al., 2021a;Knezevic et al., 2021a;Gojkovic et al., 2021b;Knezevic et al., 2021b;Strbe et al., 2021), recordings were made in deeply anesthetized rats with a cannula (BD Neoflon ™ Cannula) connected to a pressure transducer (78534C MONITOR/TERMINAL; Hewlett Packard, United States), inserted into the portal vein, inferior caval vein, and superior sagittal sinus, as well as the abdominal aorta at the level of the bifurcation at 15, 30, 60, or 120 min ACS-time. For superior sagittal sinus pressure recording, we made a single burr hole in the rostral part of the sagittal suture, above the superior sagittal sinus, and cannulated the superior sagittal sinus anterior part using a Braun intravenous cannula; then, we laparatomized the rat for portal vein, inferior vena cava, and abdominal aorta pressure recording.

ECG Recording
ECGs were recorded continuously in deeply anesthetized rats for all three main leads, by positioning stainless steel electrodes on all four limbs using an ECG monitor with a 2090 programmer (Medtronic, United States) connected to a Waverunner LT342 digital oscilloscope (LeCroy, United States) at 30 min ligation time. This arrangement enabled precise recordings, measurements, and analysis of ECG parameters (Vukojevic et al., 2018;Gojkovic et al., 2020;Kolovrat et al., 2020;Gojkovic et al., 2021a;Knezevic et al., 2021a;Knezevic et al., 2021a;Gojkovic et al., 2021b;Knezevic et al., 2021b;Strbe et al., 2021). The time until extreme bradycardia and asystole was assessed.

Brain Volume and Vessel Presentation
Brain volume and vessel presentation were proportional to the change in the brain or vessel surface area. The presentation of the brain and peripheral vessels (superior mesenteric vein, portal vein, inferior caval vein, azygos vein, and abdominal aorta) was recorded in deeply anesthetized rats, with a camera attached to a VMS-004 Discovery Deluxe USB microscope (Veho, United States) Knezevic et al., 2021a;Knezevic et al., 2021a;Gojkovic et al., 2021b;Knezevic et al., 2021b;Strbe et al., 2021). The border of the brain in the image was marked using ImageJ software and then the surface area of the brain was measured. This was done with brain images for both the control (saline) group and treated (BPC 157) group of rats at same intervals after the application and at the time of sacrifice. The arithmetic mean of the surface areas was calculated for both groups. Then, the ratio of these two areas was calculated as ( Acon A bpc ), where A con is the arithmetic mean brain area of the control group and A bpc is the arithmetic mean brain area of the treated group. Starting from the square-cube law equations [1] [2], an equation for the change in brain volume proportional to the change in brain surface area [6] was derived. In expressions [1][2][3][4][5], l is defined as any arbitrary one-dimensional length of the brain (for example, rostrocaudal length of the brain), used only for defining the one-dimensional proportion (l 2 /l 1 ) between two observed brains and as an inter-factor (and because of that not measured [6]) for deriving final expression [6]. The procedure was as follows:

Liver and Spleen Weights
Liver and spleen weights are expressed as a percentage of total body weight (for normal rats, liver, 3.2-4.0%; spleen, 0.20-0.26%).

MICROSCOPY
From rats, at end of the experiment, the brain, liver, kidney, stomach, duodenum, jejunum, colon, rectum, lungs, and heart were fixed in 10% neutral buffered formalin (pH 7.4) at room temperature for 24 h. Representative tissue specimens were embedded in paraffin, sectioned at 4 μm, stained with hematoxylin and eosin (H&E), and evaluated by light microscopy using an Olympus 71 digital camera and an Olympus BX51 microscope (Japan) acquiring digital images saved as uncompressed 24-bit RGB TIFF files.
Lung histology. A scoring system was used to grade the degree of lung injury in lung tissue analysis Knezevic et al., 2021a;Knezevic et al., 2021a;Gojkovic et al., 2021b;Knezevic et al., 2021b). Features included focal thickening of the alveolar membranes, congestion, pulmonary edema, intraalveolar hemorrhage, interstitial neutrophil infiltration, and intra-alveolar neutrophil infiltration. Each feature was assigned a score from 0 to 3 based on its absence (0) or presence to a mild (1), moderate (2), or severe (3) degree, and a final histology score was determined (Murao et al., 2003).
Gastrointestinal histology. As previously described Kolovrat et al., 2020;Gojkovic et al., 2021a;Knezevic et al., 2021a;Knezevic et al., 2021a;Gojkovic et al., 2021b;Knezevic et al., 2021b;Strbe et al., 2021), intestinal tissue damage was analyzed using a histologic scoring scale adapted from Chui and coworkers (Chui et al., 1970) on a scale of 0-5 (normal to severe) in three categories (mucosal injury, inflammation, and hyperemia/hemorrhage) for a total score of 0-15, as described by Lane and coworkers (Lane et al., 1997). Morphologic features of mucosal injury were based on different grades of epithelial lifting, villi denudation, and necrosis; grades of inflammation were graded from focal to diffuse according to lamina propria infiltration or subendothelial infiltration; hyperemia/hemorrhage was graded from focal to diffuse according to lamina propria or subendothelial localization. In addition, the villi height was assessed as well (normal villi height as indicated before Teshfam et al., 2010)).

Statistical Analysis
Statistical analysis was performed by parametric one-way analysis of variance (ANOVA), with the Newman-Keuls post hoc test or the non-parametric Kruskal-Wallis test and subsequently the Mann-Whitney U test to compare groups. Values are presented as the mean ± standard deviation (SD) and as the minimum/ median/maximum. To compare the frequency difference between groups, the chi-squared test or Fischer's exact test was used. p < 0.05 was considered statistically significant.

RESULTS
We revealed that, despite permanently increased intraabdominal hypertension (grade III and grade IV), a perilous syndrome occurred peripherally and centrally, the reversal of the abdominal compartment syndrome induced by the stable gastric pentadecapeptide BPC 157 application was quite consistent. With sustained increased intra-abdominal pressures and pentadecapeptide BPC 157 application, otherwise imminent abdominal compartment syndrome (i.e., 25 mmHg or 30 mmHg, or 40 mmHg or 50 mmHg for 25, 30, and 60 min (thiopental) and for 120 min (esketamine)) did not appear. This was seen with the portal, caval, aortal, and superior sagittal sinus pressure assessment, reduced major ECG disturbances, nearly abrogated arterial and vein thrombosis, and preserved presentation of the brain, heart, lungs, liver, kidneys, and gastrointestinal tract, with no lethal outcomes despite the permanent maintenance of high intraabdominal pressure. Both BPC 157 regimens (µg and ng) provided a similar therapeutic effect in all of the investigated protocols of abdominal compartment syndrome.
Commonly, all increased intra-abdominal pressures (i.e., 25, 30, 40, and 50 mmHg) produced a highly noxious syndrome, which occurred both peripherally and centrally. This noxious syndrome resembled the major vessel occlusion-induced syndromes (Vukojevic et al., 2018;Gojkovic et al., 2020;Kolovrat et al., 2020;Gojkovic et al., 2021a;Knezevic et al., 2021a;Knezevic et al., 2021a;Knezevic et al., 2021b) or "occlusion-like" syndromes that appear after intragastric Frontiers in Pharmacology | www.frontiersin.org December 2021 | Volume 12 | Article 718147 application of absolute alcohol  and intraperitoneal application of lithium overdose (Strbe et al., 2021), in particular, similar to the acute Budd-Chiari syndrome and acute suprahepatic inferior caval vein occlusion . Contrarily, in rats with high intraabdominal pressure, the application of BPC 157 had a considerable therapeutic effect. For this effect, in all BPC 157treated rats, the common key finding may be the rapidly activated azygos vein collateral pathway, which combined the inferior caval vein and left superior caval vein, to reverse the rapid presentation of this deadly syndrome.

Blood Pressure Disturbances
Perceived as a cause-consequence relation, the important evidence is that BPC 157 reduced blood pressure disturbances that were induced by increased intra-abdominal pressures, shown to be quite severe and noted peripherally (portal and caval hypertension, aortal hypotension) as well centrally (superior sagittal sinus hypertension) ( Figure 1). The severely increased pressure values in the portal vein, inferior caval vein, and superior sagittal sinus, as well as the decreased pressure values in the abdominal aorta, were markedly attenuated with BPC 157 application.

Collateral Pathways, Blood Vessels, and Brain Gross Presentation
As a follow-up to the attenuation of blood pressure disturbances, peripherally and centrally, there was a reduction in blood stasis by activating the collateral pathway to compensate for major vessel occlusion due to mechanical compression. Consequently, there were particular effects of BPC 157 on the relative volume of the vessels and FIGURE 1 | Blood pressure, mmHg (in the superior sagittal sinus (SSS), portal vein (PV), abdominal aorta (AA), inferior caval vein (ICV)), in the thiopentalanesthetized rats with the increased intra-abdominal pressures at 50 mmHg for 25 min, at 30 mmHg or 40 mmHg for 30 min, and at 25 mmHg for 60 min increased intra-abdominal pressures-time, and in the esketamine-anesthetized rats with the increased intra-abdominal pressures at the 25 mmHg for 120 min increased intraabdominal pressures-time, following medication (BPC 157 10 μg/kg (light gray bars), 10 ng/kg (dark gray bars); saline 5 ml/kg (white bars)) given subcutaneously at 10 min increased intra-abdominal pressures-time. Means ± SD, *P˂0.05, vs. control.
Frontiers in Pharmacology | www.frontiersin.org December 2021 | Volume 12 | Article 718147 FIGURE 2 | Relative brain and vessels volume (volume control/volume BPC 157, %) in the thiopental-anesthetized rats with the increased intra-abdominal pressures at 50 mmHg for 25 min, at 30 mmHg or 40 mmHg for 30 min, and at 25 mmHg for 60 min increased intra-abdominal pressures-time, and in the esketamineanesthetized rats with the increased intra-abdominal pressures at 25 mmHg for 120 min increased intra-abdominal pressures-time, following medication (BPC 157 10 μg/kg (light gray bars), 10 ng/kg (dark gray bars); saline 5 ml/kg (not shown, control/control as control, 100% for comparison)) given subcutaneously at 10 min increased intra-abdominal pressures-time. Means ± SD, *P˂0.05, vs. control.
Frontiers in Pharmacology | www.frontiersin.org December 2021 | Volume 12 | Article 718147 8 brain that may be indicative of the activated defensive response (Figures 2, 3, 4, 5). BPC 157 may decrease the relative volume of the superior mesenteric vein and inferior caval vein and brain ( Figures  2, 4, 5). These veins appeared congested (Figures 3, 4), likely due to failed vessels and trapped blood volume (note that the liver and spleen relative weights were increased, along with hemorrhagic lesions in the stomach) (Figures 9, 10) (Figures 3, 4, 5). Evidently, as a particular effect on blood vessels, congestion was reduced by activating the collateral bridging pathway, i.e., the azygos vein (Figure 2), as BPC 157 increased the azygos vein relative volume (Figures 2, 4). In this way, BPC 157 combined the inferior caval vein and left superior caval vein to reestablish blood flow. Finally, regarding brain swelling and increased volume (associated with considerable brain injuries) (Figures 2, 5), BPC 157 rapidly induced a considerable decrease toward normal brain presentation (Figures 2, 5).

Thrombosis
Likewise, in the cause-consequence course of the therapy, BPC 157 reduced thrombosis, both peripherally and centrally. Without therapy, thrombosis imminently occurred along with high intra-abdominal pressure, peripherally in veins (i.e., portal vein and inferior caval vein, superior mesenteric vein, hepatic veins, and external jugular vein) and in arteries (i.e., superior mesenteric artery, hepatic artery and abdominal aorta) and centrally (i.e., superior sagittal sinus) ( Figure 6). Note that, without therapy, while thrombosis was present in all investigated vessels, with an initial increase of 25 mm, the most prominent clots appeared in the hepatic veins. With further pressure increases (30, 40, and 50 mmHg), clot formation generally increased, and prominent clots also appeared in the portal vein and inferior caval vein and in the abdominal aorta.

Heart and ECG Disturbances
Commonly, high intra-abdominal pressures were timely along with the nodal rhythm, with dominant ST-elevation and bradycardia. Extreme bradycardia and asystole appeared as the ultimate outcome, at 20 ± 2 min (50 mmHg), 25 ± 5 min and 28 ± 2 min (30 mmHg and 40 mmHg), and 55 ± 8 min (25 mmHg) in control rats under thiopental anesthesia and at 110 ± 25 min in esketamine-anesthetized control rats. However, the evidence shows that despite continuously maintaining high intraabdominal pressure, in all BPC 157-treated rats, heart function was consistently maintained, with fewer ECG disturbances. The sinus rhythm was preserved, with occasional first-degree AV block, but with no ST-elevation. Extreme bradycardia and asystole were not observed. This occurred along with normal heart microscopic presentation, unlike the myocardial congestion and sub-endocardial infarction observed in controls ( Figure 11).

Gastrointestinal, Lung, Liver, Kidney, and Heart Lesions
Consequently, as part of the cause-consequence therapeutic course, i.e., reduced intracranial (superior sagittal sinus), portal, and caval hypertension, reduced aortal hypotension, and activated collateral pathway, BPC 157 reduced the severity of lesions in the gastrointestinal tract and other organs commonly noted in the untreated rats with high intra-abdominal pressures (Figures 7,8,9,10,11;Supplementary Figures S1,S2).
With an increase in severity from the upper toward the lower part of the gastrointestinal tract, control rats demonstrated transmural hyperemia of the entire gastrointestinal tract, stomach, duodenum, and small and large bowel wall, along with a reduction in the villi in the intestinal mucosa, crypt reduction with focal denudation of superficial epithelia, and dilatation of the large bowel (Figures 7,8,9,10,11;Supplementary Figures S1,S2). Regularly, in BPC 157-treated rats, we noted no or minimal congestion in the gastrointestinal mucosa with well-preserved intestinal villi and colonic crypts with no dilatation of the large bowel. Considering intraabdominal hypertension at grade III and grade IV and the therapeutic effect, it was not surprising to find a considerable decrease in villi height in all control rats with high intraabdominal pressure (Figures 7, 9; Supplementary Figures S1, S2) and preserved villi height in the BPC 157-treated rats (similar to the villi height in healthy rats, indicating preserved intestinal function despite high intra-abdominal pressure).
Without therapy, severe lesions were observed in the rats with high intra-abdominal pressures, characterized by marked congestion of the myocardium and subendocardial infarcts (Figure 11), marked congestion and large areas of intraalveolar hemorrhage in the lung (Figure 10), vascular dilation of the liver parenchyma (Figure 10), and renal congestion ( Figure 11). In contrast, as a result of treatment, the equally high intra-abdominal pressures in BPC 157-treated rats led to only mild congestion in the gastrointestinal tract, liver, and kidney (Figures 7, 8, 9, 10, 11), particularly with high intra-  abdominal pressures at 40 and 50 mmHg (otherwise, no changes in the liver and renal parenchyma were observed). The myocardium was preserved, with no change in the lung parenchyma ( Figure 8, 10, 11).
In general, congestion of the cerebral and cerebellar cortex, hypothalamus/thalamus, and hippocampus was observed, with edema and large areas with increased numbers of karyopyknotic cells, as well as intracerebral hemorrhage, mostly in the infratentorial space, affecting the cerebello angle/area (Figures 12, 13, 14, 15). We noted an increased number of karyopyknotic cells in all four regions, i.e., the cerebral and cerebellar cortex, hippocampus, and hypothalamus/thalamus ( Figure 14). Especially, there was karyopyknosis and degeneration of Purkinje cells of the cerebellar cortex and marked karyopyknosis of pyramidal cells in the hippocampus. In particular, these brain lesions appeared to be distinctively affected by high intra-abdominal pressure; i.e., the most progressive hippocampal neuronal damage was found with the highest intra-abdominal pressure. Contrarily, as a cause-consequence of BPC 157 therapy, i.e., reduced intracranial (superior sagittal sinus) hypertension and gross brain swelling, along with reduced portal and caval hypertension, aortal hypotension, abrogated thrombosis, and an activated collateral pathway, these lesions were largely reduced in BPC 157-treated rats, with a highly protected cortex, hypothalamus/thalamus, and hippocampus, as well as healthy Purkinje cells in the Quantitative analysis of neuronal damage in the karyopyknotic areas in all four neuroanatomic structures showed no or only a few karyopyknotic neural cells FIGURE 6 | Thrombus mass, g (in the superior sagittal sinus (SSS), portal vein (PV), inferior caval vein (ICV), superior mesenteric vein (SMV), external jugular vein (EJV), hepatic veins (HV), abdominal aorta (AA), superior mesenteric artery (SMA) and hepatic artery (HA)) in the thiopental-anesthetized rats with the increased intraabdominal pressures at 50 mmHg for 25 min, at 30 mmHg or 40 mmHg for 30 min, at 25 mmHg for 60 min increased intra-abdominal pressures-time and in the esketamine-anesthetized rats with the increased intra-abdominal pressures at 25 mmHg for 120 min increased intra-abdominal pressures-time, following medication (BPC 157 10 μg/kg (light gray bars), 10 ng/kg (dark gray bars); saline 5 ml/kg (white bars)) given subcutaneously at 10 min increased intra-abdominal pressures-time. Means ± SD, *P˂0.05, vs. control.
Frontiers in Pharmacology | www.frontiersin.org December 2021 | Volume 12 | Article 718147 12 ( Figure 12). The white matter was more vulnerable to chronic cerebral injury. No white matter lesions were found in both groups of animals using modified Bielschowsky silver staining and Klüver-Barrera staining.
In summary, after BPC 157 therapy, rats with high intraabdominal pressures (grade III and grade IV) exhibited markedly attenuated portal and caval hypertension, ameliorated aortal hypotension, and markedly attenuated superior sagittal sinus hypertension. Additionally, venous and arterial thrombosis was attenuated, both peripherally and centrally, which markedly mitigated stasis and moreover reduced brain, heart, lung, liver, kidney, and gastrointestinal lesions as the untreated result. These reductions were ascribed to the key finding of an activated particular collateral pathway, i.e., the azygos vein, which combined the inferior caval vein and left superior vein to reorganize blood flow.
With the applied procedure (i.e., 25, 30, 40, or 50 mmHg intraabdominal hypertension), there was a regular downhill chain of events, regardless of the type of anesthesia (i.e., esketamine, as ketamine is an antioxidant (Xingwei et al., 2014) that may provide a more prolonged survival period than thiopental). The abdominal wall compliance threshold was crossed mechanically, with no further stretch of the abdomen; this FIGURE 7 | Gastrointestinal lesions microscopy scoring (0-15), stomach, duodenum, jejunum, ascending colon and intestinal villi high, µm, and stomach lesions (sum of longest lesions diameters, mm), relative liver weight (% of total body weight), relative spleen weight (% of total body weight) in the thiopental-anesthetized rats with the increased intra-abdominal pressures at 50 mmHg for 25 min, at 30 mmHg or 40 mmHg for 30 min, at 25 mmHg for 60 min increased intra-abdominal pressurestime, and in the esketamine-anesthetized rats with the increased intra-abdominal pressures at 25 mmHg for 120 min increased intra-abdominal pressures-time, following medication (BPC 157 10 μg/kg (light gray bars), 10 ng/kg (dark gray bars); saline 5 ml/kg (white bars)) given subcutaneously at 10 min increased intraabdominal pressures-time. Minimum (min), maximum (max), median (med), means ± SD, *P˂0.05, vs. control.
To reverse abdominal compartment syndrome as a multiple occlusion syndrome disaster, we improved the function of the venous system with the stable gastric pentadecapeptide BPC 157. Considering the multitude of vessels that had been directly compressed, this improvement should be greater than that in specific vessel occlusion syndromes (Vukojevic et al., 2018;Gojkovic et al., 2020;Kolovrat et al., 2020;Gojkovic et al., 2021a;Knezevic et al., 2021a;Knezevic et al., 2021a;Knezevic et al., 2021b) or with an intragastric application of absolute alcohol and intraperitoneal application of lithium overdose, which induce an "occlusion-like" syndrome Strbe et al., 2021). This abdominal compartment syndrome therapy addresses more than one known initial target, i.e., single vessel occlusion (ligation) (Vukojevic et al., 2018;Gojkovic et al., 2020;Kolovrat et al., 2020;Gojkovic et al., 2021a;Knezevic et al., 2021a;Knezevic et al., 2021a;Knezevic et al., 2021b) vs. intragastric application of absolute alcohol  and intraperitoneal application of lithium overdose (Strbe et al., 2021) vs. all vessels compressed (increased intra-abdominal hypertension). Thus, by resolving and compensating for damaged functions, the reversal of the chain of harmful consequences of high intra-abdominal pressure can be achieved and abdominal compartment syndrome recovery FIGURE 9 | Illustrative presentation of gross and microscopic presentation. Gross presentation. Stomach (a, A) and liver (b,B) (white letters) after the increased intraabdominal pressure and medication (sc) (saline (5 ml/kg, left, stomach and duodenum with multiple small hemorrhagic lesions (a), and congested liver (b) presentation) or BPC 157 (10 ng/kg, right, stomach and duodenum, and liver A, B): 25 mmHg (30 min) (a, A), and 40 mmHg (30 min) (b, B). The camera attached to a VMS-004 Discovery Deluxe USB microscope (Veho, United States). Microscopy presentation. Stomach (a, A) and colon (b, B) (black letters) presentation in rats with the increased intra-abdominal pressure at 50 mmHg for 25 min treated at 10 min increased intra-abdominal pressure time with saline (control, a, b) or BPC 157 (A, B). The control group showed marked hyperemia and congestion of the stomach wall (a) and a reduction of the colonic crypts with focal denudation of the superficial epithelia (b). BPC 157-treated rats exhibit presentation close to normal gastrointestinal tract presentation (A, B). (HE; a, A, magnification ×100, scale bar 200 μm; b, B, magnification ×200, scale bar 100 μm).
Frontiers in Pharmacology | www.frontiersin.org December 2021 | Volume 12 | Article 718147 can occur. Thus, the beneficial findings in rats with severely increased intra-abdominal pressure given the stable gastric pentadecapeptide BPC 157 (for review, see Sikiric et al., 2018) likely occurred due to the effect on compressed essential vessel tributaries, both arterial and venous, peripherally and centrally. As a likely rescue pathway, as seen in the rat Budd-Chiari syndrome , superior sagittal sinus occlusion syndrome , and intragastric application of absolute alcohol  or intraperitoneal application of lithium overdose (Strbe et al., 2021), we identified the activated azygos vein pathway and the inferior vena cava-azygos vein-left superior vena cava pathway. The azygos vein pathway was fully activated in BPC 157-treated rats (and thereby provided additional direct blood flow delivery), while it was collapsed in control saline-treated rats with intraabdominal hypertension.
There may be, however, other activated bypassing loops (Vukojevic et al., 2018;Gojkovic et al., 2020;Kolovrat et al., 2020;Gojkovic et al., 2021a;Knezevic et al., 2021a;Knezevic et al., 2021a;Gojkovic et al., 2021b;Knezevic et al., 2021b). With the harmful effects of intra-abdominal hypertension, peripherally but also centrally, rats with an occluded superior sagittal sinus may be an illustrative example . Therefore, we identified central shunts through the ophthalmic vein, angularis vein, facial anterior and posterior veins, and facial vein, as well as the superior cerebral veins, the superior and inferior sinus cavernosus, the sinus petrosus, the sinus transversus, the external jugular vein, the subclavian vein, and the superior vena cava . Moreover, with BPC 157 therapy delivered topically to the swollen brain, intraperitoneally or intragastrically, a rapid attenuation of brain swelling was observed . A similar syndrome also FIGURE 10 | Lung (a, A, b, B) and liver (c, C, d, D) presentation in rats with the increased intra-abdominal pressure at 25 mmHg for 60 min (a, A, c, C) or at 50 mmHg for 25 min (b, B, d, D), treated at 10 min increased intra-abdominal pressure time with saline (control, a, b, c, d) or BPC 157 (A, B, C, D). a, b. Lung parenchyma with marked congestion and large areas of intra-alveolar hemorrhage in control rats. A, B. Normal lung parenchyma in BPC 157-treated rats. c, d. Vascular dilatation of liver parenchyma in controls, normal architecture in BPC 157 treated rats (C) and slight congestion of liver parenchyma (D). (HE; magnification ×200, scale bar 100 μm (a, A, b, B); magnification ×100, scale bar 500 μm (c, C, d, D)).
Frontiers in Pharmacology | www.frontiersin.org December 2021 | Volume 12 | Article 718147 Gojkovic et al., 2021a;Knezevic et al., 2021a;Knezevic et al., 2021a;Gojkovic et al., 2021b;Knezevic et al., 2021b;Strbe et al., 2021), this activated alternative blood flow was provided continuously maintained heart function, leading to nearnormal lung, liver, and kidney presentation, unlike the extreme congestion and hemorrhage observed in control rats. Collectively, these findings implicate that the heart, lungs, liver, and kidney are BPC 157 therapeutic targets. Thus, despite increased intra-abdominal pressure, BPC 157 therapy normalized portal and caval pressure and aortal pressure, as well as portal vein and inferior caval vein and aorta presentation. This maintenance may be essentially important. Otherwise, high portal and caval hypertension, aortal hypotension, exaggerated congestion of both the inferior caval and superior mesenteric veins, and a narrowed aorta all appear along with the most severe organ lesions. This clear damage has also been seen in other vessel occlusion studies (Vukojevic et al., 2018;Gojkovic et al., 2020;Kolovrat et al., 2020;Gojkovic et al., 2021a;Knezevic et al., 2021a;Knezevic et al., 2021a;Gojkovic et al., 2021b;Knezevic et al., 2021b;Strbe et al., 2021). Conceptually, the gastrointestinal, liver, and kidney lesions described here are illustrative cause-consequence relationships indicative of an uninterrupted injurious course. Vice versa, when the lesions are absent/abrogated, they clearly illustrate the therapeutic effect of BPC 157 and an interrupted injurious course.
Thus, specific conceptual support in rats with high intraabdominal pressures is provided by gastrointestinal tract failure, hemorrhagic lesions in the stomach, transmural hyperemia of the entire gastrointestinal tract, stomach, duodenum, and small and large bowel wall. The reduction of FIGURE 13 | Neuropathological changes of the cerebral cortex (a, A, b, B), cerebellar cortex (c, C) and pons (d, D) in rats with the increased intra-abdominal pressure at 25 mmHg for 60 min (a, A, c, C) or at 50 mmHg for 25 min (b, B, d, D), treated at 10 min increased intraabdominal pressure time with saline (control, a, b, c, d) or BPC 157 (A, B, C, D). Generalized edema and congestion (a, b, c, d) with an increased number of karyopyknotic cells were found in the cerebral cortex (a, b) that was significantly different from the cortex area in BPC 157-treated rats (A, B). In control rats, intracerebral hemorrhage was found in infratentorial space (d), mostly in cerebellopontine angle/area (c) with generalized edema and congestion of central nervous system, while no hemorrhage (C) and only mild edema was found in treated animals, mostly at 50 mmHg intra-abdominal pressure (D). (HE; magnification ×200, scale bar 100 μm (a, A, b, B, d, D); magnification ×100, scale bar 200 μm (c, C)).
Frontiers in Pharmacology | www.frontiersin.org December 2021 | Volume 12 | Article 718147 villi in the intestinal mucosa and crypt reduction with focal denudation of superficial epithelia and dilatation of the large bowel illustrate vascular failure (Chan et al., 2014). Accordingly, the liver and the kidney exhibited huge vascular congestion. Vice versa, the normalized portal and caval pressure and aortal pressure as a cause-consequence are convincing evidence of the functioning "bypassing key" (i.e., the azygos vein). Consequently, BPC 157-treated rats exhibited no or minimal congestion in the gastrointestinal mucosa, with well-preserved intestinal villi and colonic crypts and no dilatation of the large bowel, as well as a maintained vascular supply and reduced vascular failure (Chan et al., 2014). In the liver and kidney, only mild congestion was observed at the highest intraabdominal pressures. Furthermore, high intra-abdominal pressures/increased intracranial pressures led to the severe presentation of brain lesions. Equally, with therapy, the reversed injury course (increased intra-abdominal pressure/reduced intracranial FIGURE 14 | Bielschowsky and Klüver-Barrera histochemical staining presenting neuropathological changes of cerebral cortex in rats with the increased intraabdominal pressure at 30 mmHg for 30 min (a, A, b, B) treated at 10 min increased intraabdominal pressure time with saline (control a, b) or BPC 157 (A, B). In control rats, an increased number of karyopyknotic cells was found in the cerebral cortex (white arrows) (A, B) that was significantly different from the cortex area in BPC 157treated rats (a, b). (Bielschowsky staining (a, A); Klüver-Barrera staining (b, B); magnification ×600, scale bar 50 μm). Neuropathological changes of hypothalamic/ thalamic area (c, C, d, D) presentation in rats with the increased intra-abdominal pressure at 25 mmHg for 60 min (c, C) or at 50 mmHg for 25 min (d, D), treated at 10 min increased intra-abdominal pressure time with saline (control, c, d) or BPC 157 (C, D). A marked karyopyknosis was found in all control rats (marked in oval) (c, 25 mmHg/ 60 min); d, 50 mmHg/25 min) while preserved brain tissue was found in BPC 157-treated rats (C, 25 mmHg/60 min); D, 50 mmHg/25 min). (HE; magnification ×400, scale bar 50 μm).
Frontiers in Pharmacology | www.frontiersin.org December 2021 | Volume 12 | Article 718147 20 hypertension) led to reduced intracranial hypertension as the ultimate therapeutic outcome when the venous system was supported (i.e., activation of the azygos shunt). This was key in the brain as well, as pressures were not rapidly transmitted up through the venous system, and thereby brain presentation was preserved. The brain was preserved both grossly (absent brain swelling) and microscopically (consistent beneficial effect in all brain areas). Evidently, the beneficial effect of BPC 157 acted against the full range of brain lesions, in the order cerebellum cortex > hypothalamus/thalamus > cerebral cortex. The cerebellar cortex appeared to be the most affected, and the cerebral cortex was the least affected. The hippocampus, with increased lesion severity at higher intra-abdominal pressures, may be seen as a particular target. On the other hand, the vicious course induced by high intra-abdominal pressure can be simultaneously initiated and perpetuated from different sites (it should be noted that intracranial hypertension may essentially cause pulmonary edema and impair pulmonary circulation (Chen, 2009)).
Both BPC 157 regimens (µg and ng) had a similar therapeutic effect in all of the investigated protocols of abdominal compartment syndrome. Further cause-consequence evidence could be seen in BPC 157-treated rats with high intraabdominal pressures, as treatment largely abrogated both arterial and venous thrombosis. This was seen before with vessel occlusion (Vukojevic et al., 2018;Gojkovic et al., 2020;Kolovrat et al., 2020;Gojkovic et al., 2021a;Knezevic et al., 2021a;Knezevic et al., 2021a;Knezevic et al., 2021b), alcohol and lithium intoxication Strbe et al., 2021), and abdominal aorta anastomosis (Hrelec et al., 2009). The effect FIGURE 15 | Neuropathological changes of cerebellar cortex (a, A, b, B) and hippocampus (c, C, d, D) in rats with the increased intra-abdominal pressure at 25 mmHg for 60 min (a, A, c, C) or at 50 mmHg for 25 min (b, B, d, D), treated at 10 min increased intra-abdominal pressure time with saline (control, a, b, c, d) or BPC 157 (A, B, C, D). Control rats exhibited within cerebellar area karyopyknosis and degeneration of Purkinje cells (a, b). Marked and progressive karyopyknosis and degeneration of pyramidal cell of the hippocampus was observed in control rats (arrows) at 25 mmHg intraabdominal pressure (c) and even more at 50 mmHg intra-abdominal pressure (d). No change was found in the cerebellar and hippocampal area in BPC 157-treated rats at 25 mmHg intra-abdominal pressure (A, B, C) and only rare hippocampal karyopyknotic cells (arrows) at 50 mmHg intra-abdominal pressure (D) (HE; magnification ×400, scale bar 50 μm).
Finally, calvariectomy and/or laparotomy, used in therapy to reduce abdominal compartment syndrome (Hunter and Damani, 2004;Hedenstierna and Larsson, 2012), and in the present study to assess intracranial (superior sagittal sinus), portal, inferior caval vein, and aortal pressure, and brain, organ, and vessel presentation, may not interfere with the worst circumstances created in the abdominal compartment syndrome. In fact, the evidence shows that superior sagittal sinus hypertension even increased slightly after laparotomy. Thereby, the evidenced severe superior sagittal sinus, portal, and caval hypertension and aortal hypotension occurred along with the rapid worsening that would appear along with decompression (Hsu et al., 2004). The reduction with BPC 157 is along with its previous reducing potential on severe superior sagittal sinus, portal, and caval hypertension and aortal hypotension (Vukojevic et al., 2018;Gojkovic et al., 2020;Kolovrat et al., 2020;Gojkovic et al., 2021a;Knezevic et al., 2021a;Knezevic et al., 2021a;Gojkovic et al., 2021b;Knezevic et al., 2021b;Strbe et al., 2021).
In conclusion, these findings related to BPC 157 therapy may be important in both shorter and more prolonged periods of abdominal compartment syndrome development and reduction. Of note, intra-abdominal hypertension is quite frequent in critically ill patients and the cause of multiorgan dysfunction (Hunter and Damani, 2004;Hedenstierna and Larsson, 2012). Also, we should acknowledge that animal models although quite different (Schachtrupp et al., 2007) (here, 25, 30, 40, and 50 mm Hg by intraperitoneal insufflation of ordinary air controlled and maintained by a manual manometer leads to invariable abdominal compartment syndrome), correlate fairly well with the circumstances in humans. Therefore, in principle, the application of pentadecapeptide BPC 157 therapy is effective in particular venous occlusion syndromes, as well as for recovery from all compressed blood vessels and the consequent syndrome (Vukojevic et al., 2018;Gojkovic et al., 2020;Kolovrat et al., 2020;Gojkovic et al., 2021a;Knezevic et al., 2021a;Knezevic et al., 2021a;Gojkovic et al., 2021b;Knezevic et al., 2021b;Strbe et al., 2021). Fully achieved reduction of severe lesions in the brain, heart, lungs, liver, kidneys, and gastrointestinal tract reduced thrombosis in both veins and arteries, peripherally and centrally, and fully abrogated intracranial (superior sagittal sinus), portal, and caval hypertension and aortal hypotension may be regarded as a proof of concept. This study provides evidence of reductions in all the consequences of intra-abdominal hypertension, even grade III and grade IV, which may not be concerned by the relative paucity of BPC 157 clinical data Seiwerth et al., 2021;Vukojevic et al., 2022). BPC 157 has also been shown to be efficacious in ulcerative colitis (for review, see Sikiric et al., 2011;Sikiric et al., 2012;Sikiric et al., 2013;Sikiric et al., 2018;Sikiric et al., 2020b), in both the clinical setting (Veljaca et al., 2003;Ruenzi et al., 2005) and the experimental animal models (for review, see Sikiric et al., 2011;Sikiric et al., 2012;Sikiric et al., 2013;Sikiric et al., 2018;Sikiric et al., 2020b) and complications (for review, see Sikiric et al., 2020b). An important point regarding application in practice includes Frontiers in Pharmacology | www.frontiersin.org December 2021 | Volume 12 | Article 718147 various species (i.e., Tlak Gajger et al., 2018). However, the most important advantage is the very safe profile of BPC (the lethal dose (LD1) could be not achieved) (Seiwerth et al., 2018), emphasized in terms of its physiological role (assessed using in situ hybridization and immunostaining for BPC 157 in the human gastrointestinal mucosa, lung bronchial epithelium, the epidermal layer of the skin, and kidney glomeruli) (Seiwerth et al., 2018). This point was recently confirmed in a large study by Xu and collaborators . In this context, also for practical purposes, providing that the therapeutic effects speak for themselves, we provide a good background for further application of BPC 157 as a therapy.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusion of this article will be made available by the authors, without undue reservation.

ETHICS STATEMENT
The animal study was reviewed and approved the Ethics Committee of School of Medicine Zagreb.

AUTHOR CONTRIBUTIONS
MT and SG were responsible for the conceptualization. IK and SS were responsible for the methodology. IB, MK, and MS validated the results. ZM, GS, and HV conducted the formal analysis. LB, AS, and EL performed the investigation. IK, MM, and KS obtained the resources. AK, SS, and HZ contributed to the visualization. TK and IS administered the project. AB, SS, and PS wrote the original draft and reviewed and edited the work.

FUNDING
This work was supported by the University of Zagreb, Zagreb, Croatia (Grant BM 099).