The effect of pentadecapeptide BPC 157 on hippocampal ischemia/reperfusion injuries in rats

Abstract Background and purpose We focused on the, yet undescribed, therapy effect of the stable gastric pentadecapeptide BPC 157 in hippocampal ischemia/reperfusion injuries, after bilateral clamping of the common carotid arteries in rats. The background is the proven therapy effect of BPC 157 in ischemia/reperfusion injuries in different tissues. Furthermore, there is the subsequent oxidative stress counteraction, particularly when given during reperfusion. The recovering effect it has on occluded vessels, results with activation of the alternative pathways, bypassing the occlusion in deep vein thrombosis. Finally, the BPC 157 therapy benefits with its proposed role as a novel mediator of Roberts’ cytoprotection and bidirectional effects in the gut‐brain axis. Materials and Methods Male Wistar rats underwent bilateral clamping of the common carotid arteries for a 20‐min period. At 30 s thereafter, we applied medication (BPC 157 10 µg/kg; or saline) as a 1 ml bath directly to the operated area, that is, trigonum caroticum. We documented, in reperfusion, the resolution of the neuronal damages sustained in the brain, resolution of the damages reflected in memory, locomotion, and coordination disturbances, with the presentation of the particular genes expression in hippocampal tissues. Results In the operated rats, at 24 and 72 hr of the reperfusion, the therapy counteracted both early and delayed neural hippocampal damage, achieving full functional recovery (Morris water maze test, inclined beam‐walking test, lateral push test). mRNA expression studies at 1 and 24 hr, provided strongly elevated (Egr1, Akt1, Kras, Src, Foxo, Srf, Vegfr2, Nos3, and Nos1) and decreased (Nos2, Nfkb) gene expression (Mapk1 not activated), as a way how BPC 157 may act. Conclusion Together, these findings suggest that these beneficial BPC 157 effects may provide a novel therapeutic solution for stroke.


| INTRODUC TI ON
Stroke remains one of the leading causes of death and long-term disability, and the urgent quest for an effective therapeutic solution is evidenced by numerous failed clinical trials (Holloway & Gavins, 2016) but also in novel ways of trying to treat the disease (Amani, Habibey, et al., 2019;Amani, Kazerooni, Hassanpoor, Akbarzadeh, & Pazoki-Toroudi, 2019). Although ischemia is the main culprit in stroke-related injuries, another crucial part of the stroke is reperfusion, an event taking place after the ischemic area is resupplied with blood (Chamorro, Dirnagl, Urra, & Planas, 2016;Jayaraj, Azimullah, Beiram, Jalal, & Rosenberg, 2019). It is vital to re-establish blood flow, but this act also contributes to neuronal injury by activating an immunological response and leading to endotoxicity, neuroinflammation, and oxidative and nitrosative stress, thereby worsening the outcome of ischemia (Chamorro et al., 2016;Jayaraj et al., 2019).
Therefore, we focused on the yet undescribed therapeutic effect of the stable gastric pentadecapeptide BPC 157 (Kang et al., 2018;Seiwerth et al., 2014Seiwerth et al., , 2018Sikiric et al., 2010Sikiric et al., , 2011Sikiric et al., , 2012Sikiric et al., , 2013Sikiric et al., , 2014Sikiric et al., , 2016Sikiric et al., , 2017Sikiric et al., , 2018 in reperfusion of ischemia/reperfusion stroke injuries after bilateral clamping of the common carotid artery in rats. With the BPC 157 therapy administered during reperfusion, we would achieve the resolution of damage sustained in the brain, in particular in hippocampal regions CA1-4 similar to damage reflected in memory, locomotion, and coordination disturbances. Likewise, this notion will be further supported by the detection of expression of particular genes (Vegfr2,Src,Nos2,Nos1,Nos3,Akt1,Kras,Mapk1,Srf,Foxo1,Nfkb1,and Egr1) in hippocampal tissue.

| Animals
Study protocols were conducted in male Albino Wistar rats that had a body weight 200-250 g, were 12 weeks old and were bred in-house at the animal facility of the Department of Pharmacology-School of Medicine, Zagreb, Croatia. This is an animal facility registered with the Directorate of Veterinary (Reg. No: HR-POK-007).
Laboratory rats were acclimated for 5 days and randomly assigned to their respective treatment group (at least eight rats for each experimental group, depending on the method that was evaluated).
Laboratory animals were housed in PC cages in conventional labora-  Observers, who were blind to treatment regimens, assessed the experiments and evaluated the neurological tests.

| Surgery procedure and medication
Rats were anesthetized by intraperitoneal injection of thiopental (50 mg/kg) and diazepam (5 mg/kg). After anesthesia induction, each rat was placed in the supine position and fixed on the operating table. A midline incision of approximately two centimeters was made in the neck, and then both common carotid arteries and common jugular veins were exposed carefully by blunt dissection. After the vagus nerve was carefully separated from the carotid artery, cerebral ischemia was induced by bilateral clamping of the common carotid arteries. Bilateral clamping of the common carotid arteries was relieved at the end of the 20-min period. Thirty seconds later, we applied medication (BPC 157 10 µg/kg; or saline as a 1 ml bath directly on the surgical area. Five minutes after that, the incision was sutured back in layers. The sutured area was cleaned with 70% ethanol and sprayed with an antiseptic solution.

| Morris water maze test
We examined spatial learning and memory of the rats by using a spatial version of the Morris water maze (MWM) test (Vorhees & Williams, 2006). The training period was conducted through five consecutive days before the animals underwent surgery. There was a final assessment 24 hr after surgery. In each training period, the rats received four trials (with an intertrial gap of 10 min) in which the invisible platform was placed in the same location. The trial was complete once the rat found the platform and remained on the platform for 10 s or until 60 s had elapsed. To assess spatial learning, the latency time to find the invisible platform was measured for each animal. The Day 5 latency time (seconds), which was averaged from 4 trials, to locate the hidden platform in the water maze was taken as an index of acquisition or learning and a baseline measure. The difference between training latency time and postoperative latency time (ΔT) was calculated.

| Inclined beam walk test
Locomotor capabilities were evaluated using the inclined beamwalking test, as described previously . Each animal was individually placed on a 110 cm long and 1.5 cm wide wooden bar that was inclined at an angle of 60° from the ground, and the rats were given 60 s to walk the beam. The motor performance of each rat was scored (0-4) before global cerebral ischemia and 24 hr after global cerebral ischemia/reperfusion, as follows: 0completely unable to walk on the beam; 1-able to walk less than ¼ of the beam length; 2-able to walk more than ¼ but less than ½ of the beam length; 3-able to walk more than ½ but less than ¾ of the beam length; and 4-able to more than ¾ of the beam length or to walk the whole beam in 60 s.

| Lateral push test
Before stroke inducing surgery and at 24 hr after surgery, the animal was placed on a rough surface for firm grip and was evaluated for resistance to a lateral push from either side of the shoulder, as described previously (Gaur, Aggarwal, & Kumar, 2009). An animal with increased or decreased resistance to a lateral push after ischemia was assigned a + or -score, respectively (Gaur et al., 2009), and the percentage of rats showing resistance to a lateral push was recorded.

| Histopathological analysis of the hippocampus
Whole brains were fixed in 10% neutral buffered formalin for 48 hr. After fixation, coronal sections were made through the middle part of the hippocampus. Brain slabs were dehydrated in graded ethanol (70%, 80%, 96%, and 100%) and embedded in paraffin. Paraffin blocks were cut into 4-5 μm thin sections, deparaffinated in xylene, rehydrated in graded ethanol (100%, 96%, 80%, and 70%) and stained with hematoxylin and eosin. Five images were taken of the CA1 region of the hippocampus with high power (objective ×40). The healthy neurons and "red neurons" (pathological neuronal finding indicative of acute ischemic neuronal injury and subsequent apoptosis or necrosis) were manually counted and presented as an average of five images at 24 and 72 hr after surgery.

| RT-qPCR mRNA measurement
After sacrificing the animals 1 and 24 hr after reperfusion, brain tissue, that is, the hippocampus, was rapidly dissected and frozen in liquid nitrogen. The hippocampal tissue was disrupted using a T10 the range between 70% and 130% was considered biological variability (Maul, 2008;Vukojevic et al., 2018).

| Statistical analysis
Statistical analysis was performed using the Shapiro-Wilk test for distribution assessment. For parametric statistics, we used the Mann-Whitney U test to compare the difference between groups.
Additionally, for nonparametric statistics, we used Kruskal-Wallis test to compare the two groups. All statistical tests were performed using RStudio (RStudio Team, 2015). A p-value of .05 or less was considered statistically significant.

| RE SULTS
After bilateral carotid artery occlusion, significant reperfusion-induced hippocampal lesions, as well as memory and motor coordination failure, regularly appeared in tested animals. These disturbances clearly indicate our therapeutic focus.

| Neurological assessment
Morris water maze test. After surgery, latency time markedly increased in all control rats at 24 hr of reperfusion. BPC 157 completely counteracted the ischemia/reperfusion-induced damage ( Figure 1). Namely, rats that received BPC 157 during reperfusion maintained their initial latency time from before surgery.
Inclined beam walk test. All control rats at 24 hr of reperfusion exhibited the complete lack of fore and hind limb motor coordination and the inability to walk a short distance (Figure 2). Contrarily, this ischemia/reperfusion-induced defect did not appear after the BPC 157 administration. Namely, rats that received BPC 157 during reperfusion were able to walk the whole distance.
Lateral push test. At 24 hr of reperfusion in stroke rats, BPC 157-treated rats maintained an unimpaired ability to resist lateral pushes (Figure 3), unlike the controls, which all lacked resistance to lateral pushes from either side of the shoulder.

| Effect of BPC 157 on brain pathology
By manually counting all "red neurons," which are those that are pathologically altered due to ischemia and reperfusion injuries, as well as healthy neurons at 24 and 72 hr of reperfusion, we revealed that BPC 157-treated animals had far less neuronal damage than control animals and consistently had more healthy neurons (Figures 4 and 5). Thus, BPC 157 therapy seems to counteract delayed neuronal death, which consistently appears in the control of ischemia/reperfusion rats.
After the removal of the bilateral carotid artery ligation, considering the large extent of the behavioral and learning disturbances in these models, we found that functions were generally maintained after BPC 157 treatment. Notably, severely impaired locomotion capabilities including a lack of fore and hind limb motor coordination and resistance to lateral pushes from either side of the shoulder, are regular in control stroke rats (Gulati, Singh, & Muthuraman, 2014).
Thereby, with the beneficial effect in rats with either inferior caval vein occlusion (Vukojevic et al., 2018) or bilateral carotid artery occlusion, there is increased Egr1 expression, which is relevant for both, but especially so in the ischemia/reperfusion stroke rats.
This likely accommodates the essential role of Egr1 indirectly controlling the expression of other genes, and thereby, neural activity, neural plasticity, and learning (Knapska & Kaczmarek, 2004), similar to the implied role of Egr1 gene in inflammation, myocardial injury (Rayner et al., 2013), brain injuries following transient focal ischemia (Tureyen, Brooks, Bowen, Svaren, & Vemuganti, 2008) or permanent occlusion of the middle cerebral artery (Beck, Semisch, Culmsee, Plesnila, & Hatzopoulos, 2008). As a common resolving point, BPC 157 is known to simultaneously induce the expression of Egr1 and its corepressor Nab2 (Tkalcevic et al., 2007). Nab2 expression is known to be regulated by some of the stimuli that also induce Egr1 expression (Svaren et al., 1996) Activation of Akt1 is usually cytoprotective, such as its function during endothelial cell hypoxia (Somanath, Razorenova, Chen, & Byzova, 2006), and Akt1 has been extensively studied and is considered to be neuroprotective in stroke (Zhao, Sapolsky, & Steinberg, 2006), with Akt1 and Akt3 proteins degrading as early as 1 hr after stroke (Xie et al., 2013). Kras function implies an intervening capillary network, preventing high-pressure arterial blood from feeding arteries to shunt directly into the venous outflow system (Nikolaev et al., 2018). Src appears to be a critical component of NGF's function-neurite growth (Kremer et al., 1991). Src interacts directly with the NMDA receptor via its unique domain (Thomas & Brugge, 1997;Yu et al., 1996). FoxO protein activation may be required for neuronal protection (Maiese, 2015), and a fine balance in FoxO activity may be required to target cognitive loss (Maiese, 2015). The upregulation of Srf directly controls the immediate early gene response and is an essential regulator of neuronal-activity-induced gene expression (Knoll & Nordheim, 2009). Of note, most of the direct neuronal effects of VEGF-A-such as neuronal survival in cell culture models of stroke involving oxygen and glucose deprivation (Maiese, 2015) or excitotoxicity (Matsuzaki et al., 2001), hypoxic preconditioning in vitro (Wick et al., 2002), and protection in MCA occlusion models of stroke in vivo (Hayashi, Abe, & Itoyama, 1998) Namely, BPC 157 may increase and decrease and thereby control, VEGF expression during wound healing-for example, healing of a detached or transected tendon or muscle )-and BPC 157 may have a prominent angiogenic effect during healing Hsieh et al., 2017;Huang et al., 2015;Sikiric et al., 1999) while counteracting the tumor-promoting the effect of VEGF (Radeljak, Seiwerth, & Sikiric, 2004). This may be important since BPC 157 itself not only increased Vegfr2 expression in vascular endothelial cells but also immediately triggered the internalization of Vegfr2 and subsequent phosphorylation of Vegfr2, Akt, and Nos3 signaling pathways without other known ligands or shear stress (Hsieh et al., 2017). In addition, the Akt1/Nos3 signaling pathway is thought to be highly beneficial to stroke outcome, weather by upregulation of Akt1 or by subsequent activation of Nos3 (Zhou et al., 2015).
In support, BPC 157 largely interacts with the NO system in different models and species . Indicatively, BPC 157 alone may induce the release of NO in vitro in gastric mucosa from rat stomach tissue homogenates and counteract the opposite adverse effect of L-NAME (i.e., hypertension; lack of NO release in vitro) and L-arginine (i.e., hypotension; NO over-release in vitro) (Sikiric et al., 1997). In these terms, we should consider the documented triple effect of BPC 157 on NOS in the hippocampus of ischemia/reperfusion rats, which includes the in- (IL-1β) and subsequently induces a secondary inflammatory reaction and the generation of oxygen free radicals (Foncea, Carvajal, Almarza, & Leighton, 2000;Trickler, Mayhan, & Miller, 2005).

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.