Antiarrhythmic Sotalol, Occlusion/Occlusion-like Syndrome in Rats, and Stable Gastric Pentadecapeptide BPC 157 Therapy

We focused on the first demonstration that antiarrhythmics, particularly class II and class III antiarrhythmic and beta-blocker sotalol can induce severe occlusion/occlusion-like syndrome in rats. In this syndrome, as in similar syndromes with permanent occlusion of major vessels, peripheral and central, and other similar noxious procedures that severely disable endothelium function, the stable gastric pentadecapeptide BPC 157-collateral pathways activation, was a resolving therapy. After a high dose of sotalol (80 mg/kg intragastrically) in 180 min study, there were cause-consequence lesions in the brain (swelling, intracerebral hemorrhage), congestion in the heart, lung, liver, kidney, and gastrointestinal tract, severe bradycardia, and intracranial (superior sagittal sinus), portal and caval hypertension, and aortal hypotension, and widespread thrombosis, peripherally and centrally. Major vessels failed (congested inferior caval and superior mesenteric vein, collapsed azygos vein). BPC 157 therapy (10 µg, 10 ng/kg given intragastrically at 5 min or 90 min sotalol-time) effectively counteracted sotalol-occlusion/occlusion-like syndrome. In particular, eliminated were heart dilatation, and myocardial congestion affecting coronary veins and arteries, as well as myocardial vessels; eliminated were portal and caval hypertension, lung parenchyma congestion, venous and arterial thrombosis, attenuated aortal hypotension, and centrally, attenuated intracranial (superior sagittal sinus) hypertension, brain lesions and pronounced intracerebral hemorrhage. Further, BPC 157 eliminated and/or markedly attenuated liver, kidney, and gastrointestinal tract congestion and major veins congestion. Therefore, azygos vein activation and direct blood delivery were essential for particular BPC 157 effects. Thus, preventing such and similar events, and responding adequately when that event is at risk, strongly advocates for further BPC 157 therapy.

Concluding, intragastric application of the stable gastric pentadecapeptide BPC 157 (i.e., as a part of its stomach cytoprotection background [4][5][6][7]) was given shortly after intragastric application of the sotalol, or much later, in the advanced course of the sotalol bradycardias. Likely, this would show BPC 157 beneficial therapeutic effect in the counteraction of the full occlusion/ occlusion-like syndrome that may have a particular class III arrhythmic and beta-blockade background in rats.

Blood Pressure Disturbances
As the lesion spread more and more, the severity might be illustrated both peripherally (portal and caval hypertension, aortal hypotension) as well as centrally (superior sagittal sinus hypertension) ( Figure 1). Perceived as a cause-effect relation that should be essentially affected by the therapy application, BPC 157 rapidly reduced blood pressure disturbances that were induced by sotalol application, both those short-lasting (given BPC 157 application at 5 min sotalol-time) and those long-lasting (given BPC 157 application at 90 min sotalol-time). Consistently with a prominent therapy effect, both peripherally and centrally, BPC 157 application eliminated, or at least markedly attenuated, the portal and caval hypertension and intracranial (superior sagittal sinus) hypertension as well as the aortal hypotension.  Therapy included BPC 157 (10 µg/kg (light gray bars) or 10 ng/kg (dark gray bars)) or saline (5 mL/kg) (white bars). They were given as an intragastric administration at 5 min following sotalol for assessment at 15 min (e), 90 min (f ), and 180 min (g) sotalol-time. As a delayed regimen (h), the therapy was given as an intragastric administration at 90 min sotalol-time for assessment at 180 min sotalol-time. Means ± SD, * p < 0.05, at least, vs. control.

. Thrombosis
Likewise, from the very beginning, as noted already at 15 min, the cause-effect course of the therapy might be the fact that BPC 157 reduced thrombosis, both peripherally (i.e., portal vein, inferior caval vein, and abdominal aorta) and centrally (i.e., superior sagittal sinus) (Figure 1). An alike effect occurred with BPC 157 application given at 90 min sotalol-time. Without therapy, the progressed thrombosis was sustainably present in arteries and veins.

Collateral Pathways, Blood Vessels, and Brain Gross Presentation
There were increases in the relative volume of the congested vessels (superior mesenteric vein and inferior caval vein, from the trapped volume, congested liver and lung), dilated heart and collapsed vessels (azygos vein not functioning), and swollen brain ( Table 1, Figures 2-6). These might grossly appear as inactivation of the collateral pathway, inability to compensate for major vessel failure, and blood stasis observable with progressing thrombosis. Contrarily, along with attenuation/elimination of the blood pressure disturbances and thrombosis (note, congestion of the major veins, i.e., inferior caval vein and superior mesenteric vein, largely counteracted), peripherally and centrally, BPC 157 increased the azygos vein's relative volume. Thus, prompt direct blood delivery from the inferior caval vein to the left superior caval vein might occur to re-establish blood flow (heart with normal presentation). Finally, as additional proof, the brain swelling and increased volume (associated with considerable brain injuries) were rapidly counteracted by BPC 157 administration and induced a considerable decrease toward normal brain presentation. Table 1. Relative volume (control/treated) (%) of the brain, heart, azygos vein, in rats at 15 min and 90 min, and 180 min following sotalol application. Therapy included BPC 157 (10 µg/kg or 10 ng/kg) or saline (5 mL/kg). They were given as an intragastric administration at 5 min following sotalol for assessment at 15 min (period 0-15 min sotalol-time), 90 min (period 0-90 min sotalol-time) and 180 min (period 0-180 min sotalol-time). As a delayed regimen (period 90-180 min sotalol-time), the therapy was given as an intragastric administration at 90 min sotalol-time for assessment at 180 min sotalol-time. Means ± SD, * p < 0.05, at least, vs. control. Commonly, the sotalol procedure induced continuous bradycardia (Figure 7), while the prolonged PQ prolonged and QTc intervals were absent. With BPC 157 therapy, the counteraction of the sotalol-induced bradycardias might be a particular point. BPC 157 therapy might counteract bradycardias, either applied in the early or late course. However, the attenuated bradycardias persisted. This counteracting effect, providing both short-lasting bradycardias (BPC 157 given at 5 min sotalol-time) and long-lasting bradycardias (BPC 157 given at 90 min sotalol-time), occurred along with counteraction of the myocardial congestion ( Figure 8, Table 2) and dilation ( Figure 5, Table 1) and thrombosis development ( Figure 1).    post-treatment (g,H) (at 90 min and assessment at 180 min sotalol-time), saline or BPC 157 therapy given intragastrically.

Relative
. Figure 4. Illustrative presentation of the azygos vein (yellow arrows) and aorta (red arrows) in the sotalol control rats (small letters, light arrows) and in BPC 157-treated rats (capitals, dark arrows) assessed at 15 min (a,B), 90 min (c,D) and 180 min (e,F,g,H) following application of sotalol. Medication was given as an early application at 5 min after sotalol (a,B,c,D,e,F), or as delayed post-

. Heart and ECG Disturbances
Commonly, the sotalol procedure induced continuous bradycardia (Figure 7), whil the prolonged PQ prolonged and QTc intervals were absent. With BPC 157 therapy, th counteraction of the sotalol-induced bradycardias might be a particular point. BPC 15 therapy might counteract bradycardias, either applied in the early or late course However, the attenuated bradycardias persisted. This counteracting effect, providing bot short-lasting bradycardias (BPC 157 given at 5 min sotalol-time) and long-lastin bradycardias (BPC 157 given at 90 min sotalol-time), occurred along with counteractio of the myocardial congestion ( Figure 8, Table 2) and dilation ( Figure 5, Table 1) an thrombosis development ( Figure 1). Figure 6. Illustrative gross presentation of the brain in the sotalol control rats (small letters) and in BPC 157-treated rats (capitals). (i) with medication given at 5 min sotalol-time assessed at 15 min sotalol-time before sacrifice (a,B) and after sacrifice (c,D), 90 min sotalol-time before sacrifice (e,F) and after sacrifice (g,H), and 180 min sotalol-time before sacrifice (i,J) and after sacrifice (k,L). (ii) with medication given as delayed post-treatment at 90 min and assessment at 180 min sotalol-time, before sacrifice (m,N) and after sacrifice (o,P). Saline or BPC 157 therapy was given intragastrically. Pharmaceuticals 2023, 16, x FOR PEER REVIEW 13 of 38 Figure 7. ECG assessment (a,b). Heart frequency, beats/min. Therapy included BPC 157 (10 µg/kg (light gray bars) or 10 ng/kg (dark gray bars)) or saline (5 mL/kg) (white bars) intragastric application. They were given at 5 min following sotalol and assessed at 5 min thereafter, and then at 15 min intervals until the end of the experiment (180 min sotalol-time) (a). As a delayed regimen (b), the therapy was given as an intragastric administration at 90 min sotalol-time and assessed at 5 min thereafter, and then at 15 min intervals until the end of the experiment (180 min sotalol-time). Means ± SD, * p ˂ 0.05, at least, vs. control. Figure 7. ECG assessment (a,b). Heart frequency, beats/min. Therapy included BPC 157 (10 µg/kg (light gray bars) or 10 ng/kg (dark gray bars)) or saline (5 mL/kg) (white bars) intragastric application. They were given at 5 min following sotalol and assessed at 5 min thereafter, and then at 15 min intervals until the end of the experiment (180 min sotalol-time) (a). As a delayed regimen (b), the therapy was given as an intragastric administration at 90 min sotalol-time and assessed at 5 min thereafter, and then at 15 min intervals until the end of the experiment (180 min sotalol-time). Means ± SD, * p < 0.05, at least, vs. control.    Table 2. Microscopic presentation of the lesions in the heart, lung, liver, kidney, stomach, small intestine, and large intestine, and gross lesions presentation in the stomach in rats at 15 min and 90 min, and 180 min following sotalol application. Therapy included BPC 157 (10 µg/kg or 10 ng/kg) or saline (5 mL/kg). They were given as an intragastric administration at 5 min following sotalol for assessment at 15 min (period 0-15 min sotalol-time), 90 min (period 0-90 min sotalol-time) and 180 min (period 0-180 min sotalol-time). As a delayed regimen (period 90-180 min sotalol-time), the therapy was given as an intragastric administration at 90 min sotalol-time for assessment at 180 min sotalol-time. Min/Med/Max, Means ± SD, * p < 0.05, at least, vs. control.   Indicatively for a common clue that might be failed (i.e., intracranial (superior sagittal sinus), portal, and caval hypertension, and aortal hypotension, progressed thrombosis, peripherally and centrally, failed collateral recruitment), all of the sotalol regimens converge to the similar organ lesion ( Table 2, Figures 8-13). Thereby, the reduced severity of lesions by BPC 157 therapy may be seen as part of the cause-consequence therapeutic course, the recovered vascular (activation of the collateral pathway, azygos vein direct flow delivery) and heart function, along with the reduced intracranial (superior sagittal sinus), portal, and caval hypertension, and reduced aortal hypotension and immediate impact of the activated collateral pathway. In addition, in the rats challenged with sotalol, similar beneficial effects were noted with BPC 157 therapy applied intragastrically during the early or late sotalol course.

Heart Lesions
Marked myocardial congestion affecting coronary veins and arteries, as well as myocardial vessels, was consistently found in the controls at the end of all of the assessed periods ( Figure 8, Table 2).
In contrast, BPC 157 might largely counteract sotalol-course. In BPC 157-treated rats there were no changes after 15 min and 90 min sotalol-time, and only mild congestion of coronary veins at 180 min sotalol-time. In addition, a marked reversal of the lesion, even in the advanced stage, might be consistently evidenced. A delayed BPC 157 regimen, given at 90 min sotalol-time, showed no myocardial changes thereafter at 180 min sotalol-time.

Lung Lesions
Marked lung parenchyma congestion of larger blood vessels and septal capillaries consistently occurred in sotalol-control rats throughout the whole experiment ( Figure 9, Table 2). This sotalol-damaging course was largely counteracted with BPC 157 therapy. No congestion of the lung occurred at 15 min sotalol-time, while only discrete dilatation of larger blood vessels occurred at latter periods. Especially, a marked reversal of the lesion, even in the advanced stage, might occur with a delayed BPC 157 regimen, given at 90 min sotalol-time and assessed at 180 min sotalol-time.
of lesions by BPC 157 therapy may be seen as part of the cause-consequence therapeutic course, the recovered vascular (activation of the collateral pathway, azygos vein direct flow delivery) and heart function, along with the reduced intracranial (superior sagittal sinus), portal, and caval hypertension, and reduced aortal hypotension and immediate impact of the activated collateral pathway. In addition, in the rats challenged with sotalol, similar beneficial effects were noted with BPC 157 therapy applied intragastrically during the early or late sotalol course.
.  given intragastrically. Sotalol-rats treated with saline consistently exhibited a marked dilatation of larger blood vessels (black arrows) and septal capillaries net (a,c,e,g). Sotalol-rats treated with BPC 157 consistently exhibited either no dilatation of the larger blood vessels and septal capillaries (B) (period relative to therapy application 0-15 min sotalol-time) or discrete dilatation of larger blood vessels (D,F,H) (periods relative to therapy application 0-90 min, 0-180 min, 90-180 min sotalol-time).

Liver Lesions
Marked liver lesions, and marked congestion of the blood vessels in the portal tracts, sinusoids, and central veins occurred in controls at all intervals ( Figure 10, Table 2). In contrast, BPC 157 might largely counteract sotalol-course. In BPC 157-treated rats, there were no changes after 15 and 90 min sotalol-time, and only discrete dilatation of blood vessels within central veins at 180 min sotalol-time. In addition, a marked reversal of the lesion, even in the advanced stage, goes with a delayed BPC 157 regimen, given at 90 min sotalol-time, and no liver changes thereafter at 180 min sotalol-time.
min and assessment at 180 min sotalol-time) (g,H), saline (a,c,e,g) or BPC 157 therapy (B,D,F,H given intragastrically. Sotalol-rats treated with saline consistently exhibited a marked dilatation o larger blood vessels (black arrows) and septal capillaries net (a,c,e,g). Sotalol-rats treated with BP 157 consistently exhibited either no dilatation of the larger blood vessels and septal capillaries (B (period relative to therapy application 0-15 min sotalol-time) or discrete dilatation of larger bloo vessels (D,F,H) (periods relative to therapy application 0-90 min, 0-180 min, 90-180 min sotalo time). .

Kidney Lesions
Marked kidney lesions, marked dilatation, and congestion of blood vessels as well as glomerular capillary loop occurred in all controls at all intervals following sotalol ( Figure 11, Table 2). In contrast, BPC 157 might largely counteract sotalol-course. In BPC 157-treated rats given at 5 min sotalol-time, there were with no changes after 15 min, and 90 min sotaloltime, and only mild dilatation and congestion of blood vessels at 180 min sotalol-time. In addition, a marked reversal of the lesion, even in the advanced stage, goes with a delayed BPC 157 regimen, given at 90 min sotalol-time, and no kidney changes thereafter at 180 min sotalol-time.

Stomach, Small Intestine, and Colon Lesions
Without therapy, hemorrhagic stomach lesions occurred with marked congestion of submucosal blood vessels and moderate dilatation of intramucosal blood in the stomach, small intestinal and colonic wall in controls with sotalol regimen at 15 min, 90 min, 180 min, sotalol-time ( Figure 12, Table 2). In contrast, BPC 157 might largely counteract sotalolcourse ( Figure 13, Table 2). In BPC 157-treated rats, the sotalol-course occurred limited to only minor congestion of submucosal blood vessels in the stomach with no congestion of mucosal intestinal blood vessels. Thus, there was a marked reversal of the lesion development with the early application and an evident counteraction of the advanced stage with the delayed therapy application. Figure 12. Illustrative stomach (a-c), small intestine (d-f), and colon (g,h,i) congestion microscopic presentation (HE staining; magnification 100× (a-c), scale bar 200μm; magnification 200× (d-i), scale bar 200 μm) in the sotalol control rats assessed following application of sotalol, given therapy application at 15 min (a,d,g) (period 0-15 min), and at 180 min (b,e,h) (period 0-180 min) or at 180 min (c,f,i) (period 90-180 min). Saline medication was given as an early application immediately after sotalol (a,b,d,e,g,h), or as delayed post-treatment (at 90 min and assessment at 180 min sotaloltime) (c,f,i). Marked dilatation of submucosal blood vessels and moderate dilatation of intramucosal blood vessels occurred in the stomach, and small and large intestine.

Microscopic Presentation of the Brain Lesions in the Cerebrum
Thus, these results consistently revealed the beneficial effects of the BPC 157 therapy in the sotalol-induced heart failure and counteraction of the concomitant pathology, periph-erally and centrally. Counteraction of sotalol-occlusion/occlusion-like syndrome was as a whole.
Finally, there is the sotalol-occlusion/occlusion-like syndrome using a particular high sotalol protocol, 80 mg/kg intragastrically, rarely used [50], still markedly below LD50 in rats [75] as proof of the concept's applicability. In our view, this higher regimen can be more suited than the much lower dose regimens regularly focused on cardiac events.
Since the very beginning [76,77] in various species (mostly in canine models [52][53][54][55][56] but also in other species (i.e., rats, mice) [78][79][80]), regimens were mostly below 10 mg/kg as in those presented later [81,82]. These more regular doses (i.e., [83]) in regimens being accommodated to the translational human regular regimens, however, may drop even much below the requested range in humans [84,85] as translational research and drug development when back-translating the human results to rats requires dividing the rat dose by 6.2 or multiplying by 0.16 [86]. Besides, large variations in the dose regimens depending on the induced arrhythmias (i.e., 0.5 mg/kg-35 mg/kg) [87] may hamper the conclusive dosage range in animal experiments. Likewise, patients' regular 80-, 160-, and 240-mg tablets, initial dosing with 80 mg twice daily, daily dosages between 160 and 320 mg or 480 mg, may reach dosages as high as 640 mg/d [84,85]. Contrarily, the use of the consistently higher range may approach the worst circumstances, severe sotalol intoxication with suicidal attempts [88,89], and emphasize in sotalol rat research the consistently wide range of findings in the occlusion/occlusion-like syndrome [11][12][13][14][15][16][17][18][19][20][21][22]. This would illustrate the possible importance (i.e., the innate significance of sotalol bradycardias, while QTinterval prolongation may occur later [86]). Thus, we may argue resolving following the vascular and multiorgan failure for identifying and managing theoretical strengths for resolving the earliest period even in the worst circumstance (i.e., severe sotalol intoxication with suicidal attempts [88,89]). Thereby, applicable in extreme circumstances, these may be a therapy regimen likely resolving also more regular sotalol application, and circumstances and disturbances thereof. Alternatively, by dividing the rat dose [87], the original higher doses in rats are close to those used in the patients, as opposed to the lower regimens, which may be below human regimens [84,85].
Thereby, the shared vascular, endothelium lesions, and shared cardiac lesions noted with sotalol [84,85] might be analogous to these findings in the present study. There might also be a specific effect (i.e., inhibition of potassium ions efflux by sotalol-mediated blockage of the potassium channels) [90], as BPC 157 might counteract the arrhythmias and other adverse effects in a particular way. This might be seen in rats with hyperkalemia or hypokalemia and might affect potassium channels in HEK293 cells in either hyperkalemic or hypokalemic conditions [64,65].

Animals
Male Albino Wistar rats, 12-week-old, 200 g body weight, bred in-house at the Animal Pharmacology Facility, School of Medicine, Zagreb, Croatia (registered with the Veterinary Directorate (Reg. No: HR-POK-007)), randomly assigned at 6 rats/group/interval were used in all experiments. Rats were acclimated for five days and randomly assigned to their respective treatment groups; housed in polycarbonate (PC) cages (identified with dates, number of study, group, dose, number, and sex of each animal) at 20-24
Rats received therapy BPC 157 (10 µg or 10 ng/kg) or saline (5 mL/kg) (controls) as an early intragastric regimen at 5 min upon sotalol, or as delayed post-treatment, at 90 min after sotalol.

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, Minneapolis, MN, USA) connected to a Waverunner LT342 digital oscilloscope (LeCroy, Chestnut Ridge, NY, USA) (before the procedure, after sotalol and after therapy application, and before sacrifice). This arrangement enabled precise recordings, measurements, and analysis of ECG parameters (PQ intervals, QTc, heart frequency) as specifically described [8][9][10][11][12][13][14][15][16][17][18][19][20][21][22]. Specifically, therapy included BPC 157 (10 µg/kg or 10 ng/kg) or saline (5 mL/kg) intragastric application. They were given at 5 min following sotalol and assessed at 5 min thereafter, and then at 15 min intervals until the end of the experiment (180 min sotalol-time). As a delayed regimen, the therapy was given as an intragastric administration at 90 min sotalol-time and assessed at 5 min thereafter, and then at 15 min intervals until the end of the experiment (180 min sotalol-time).

Brain Volume, Heart, and Vessel Volume Presentation
We applied the procedure used in our previous vascular studies [8][9][10][11][12][13][14][15][16][17][18][19][20][21][22]. Brain volume and vessel volume and heart volume were proportional to the change in the brain or vessel or heart surface area. The presentation of the brain and peripheral vessels (superior mesenteric 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, Claymont, DE, USA). The border of the brain (or vessels, or heart) in the image was marked using ImageJ software and then the surface area of the brain (or vessels, or heart) was measured. This was done with brain (or aorta or veins) images for healthy rats, and then/or for both the control (saline) group and treated (BPC 157) group of rats at the 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 ( A con A bpc ), where Acon is the arithmetic mean brain (or veins, or aorta, or heart) area of the control group and Abpc is the arithmetic mean brain (or veins or aorta or heart) area of the treated group. Starting from the square-cube law Equations [1] and [2], an equation for the change in brain (or veins, or aorta, or heart) volume proportional to the change in brain surface area (or veins, or aorta, or heart) [6] was derived. In expressions [1]- [5], l is defined as any arbitrary one-dimensional length of the brain (for example rostro-caudal length of the brain) (or veins, or aorta, or heart), used only for defining the one-dimensional proportion (l2/l1) between two observed brains (or vessels or heart) and as an inter-factor (and because of that not measured [6]) for deriving final expression [6].
We also assessed the neuronal pathological changes in acquired digital images saved as uncompressed 24-bit RGB TIFF files in the software program AnalySIS (Olympus Soft Imaging System GmbH, Munster, Germany) performing quantitative analysis of neuronal damage in the karyopyknotic areas. The neurons of the cortical cerebral, cerebellar region, hippocampus, and hypothalamus were counted in 10 different high-powered fields (HPF, 400×) and 3 to 5 serial sections of each sample were used to do the count as described [91]. The field size was 0.24 µm 2 .
We used four criteria for the estimation of the edema: pale myelin, sieve-like appearance of myelinated areas, dilation of perivascular and pericellular spaces, and vacuolar appearance of the neuropil of gray matter. Edema was graded as heavy, moderate, slight, or no edema (score 0-3) [92].

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.
Thus, we obtained the beneficial effects of the BPC 157 therapy in the sotalol-induced heart failure and counteraction of the concomitant pathology, peripherally and centrally, counteraction of occlusion/occlusion-like syndrome as a whole. Therefore, also con-fronted with the disabled beta sympathetic system, it might be that the heart failure cause-consequence circuit might occur in a multidirectional way that BPC 157 therapy might beneficially affect as a whole.