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Pentadecapeptide BPC 157 shortens duration of tetracaine- and oxybuprocaine-induced corneal anesthesia in rats

SUMMARY We focused on the relationship of 0.5% tetracaine- and 0.4% oxybuprocaine-induced corneal anesthesia in rats, and pentadecapeptide BPC 157 (0.4 µg/eye), along with nitric oxide synthase (NOS) inhibitor N(gamma)-nitro-L-arginine methyl ester (L-NAME) (0.1 mg/eye) and/or NOS substrate L-arginine (2 mg/eye), applied in the form of eye drops. We assessed corneal sensitivity recovery (Cochet-Bonnet esthesiometer), corneal lesion elimination (staining with 10% fluorescein) and decrease in tear volume (Schirmer test). BPC 157 administration had a full counteracting effect. Recovery also occurred in the presence of NOS blockade and NOS substrate application. L-arginine eventually shortened duration of corneal insensitivity and exerted corneal lesion counteraction (and counteraction of tetracaine-induced decrease of tear volume) only in earlier but not in later period. L-NAME application led to longer duration of corneal insensitivity, increase in corneal lesions and decrease in tear volume. When L-NAME and L-arginine were applied together, they antagonized each other’s effect. These distinctions may indicate particular NOS involvement (corneal insensitivity vs. corneal lesion along with tear production), distinctively affected by the administration of NO agents. However, additional BPC 157 co-administration would re-establish counteraction over topical ophthalmic anesthetic-induced effect, be it in its early or late course. We suggest BPC 157 as an antidote to topical ophthalmic anesthetics.

indicatively, counteraction of bupivacaine arrhythmias 14 may be seen as part of the larger beneficial effect providing that BPC 157 counteracts various arrhythmias [14][15][16][17][18][19] . likewise, with BPC 157 application, counteraction of tetracaine-and oxybuprocaine-induced corneal anesthesia may appear along with healing of perforating corneal injury and total debridement of corneal epithelium, thus maintaining corneal transparency 20,21 . BPC 157 counteracts the damaging effects of lacrimal gland extirpation and dry eye syndrome in rats 22 . We also revealed nOS dependence of atropineinduced mydriasis and l-nAMe-and l-arginineinduced miosis and reversal by the pentadecapeptide BPC 157 in both rats and guinea pigs 23 . This may be particularly important considering corneal lesions that may be induced by local anesthetic application [24][25][26][27] .
On the other hand, BPC 157 produced analgesia in the MgSO4 and acetic acid test in mice, a model of prolonged pain associated with tissue injury 29 . This indicates that BPC 157 may have local anesthetic activity of its own.

Animals
Wistar Albino male rats (200 g b.w.) were randomly assigned to the experiments (6 animals at least per experimental group and interval), all of which were approved by the local ethics Committee of the School of Medicine, university of Zagreb, Zagreb, Croatia (no. 380-59-10106-19-111/100; 641-01/19-02/01). furthermore, all experiments were carried out under blind protocol and the effect was assessed by examiners who were completely unaware of the given protocol. The research and experiments also followed the ArVO Statement for the use of Animals in Ophthalmic and Vision research.

Corneal sensitivity measurement
using the Cochet-Bonnet esthesiometer (luneau Ophtalmologie, Chartres, france), a corneal sensitivity measurement device 24 , corneal sensitivity was measured in rats administered 0.5% tetracaine or 0.4% oxybuprocaine before corneal anesthesia, then after tetracaine or oxybuprocaine application, before therapy application, at 1 min, and with therapy application, at 5-minute intervals until full sensitivity was restored.
Briefly, the Cochet-Bonnet esthesiometer consists of nylon monofilament that can be pulled out and pro-duces a pressure force at its top, which is inversely proportional to the length of the elongated portion of the monofilament. The nylon filament is 0.12 mm in diameter and is 6 cm long. Corneal sensitivity testing started with gentle touch of the fully extended tip of the esthesiometer perpendicular to the cornea and applying a small force to finely distort the filament. if the rat blinked, it was recorded as a positive response. if the blinking response was absent, filament length was reduced by 1 centimeter and the procedure was repeated until a positive response was obtained. The absence of positive response when the filament length is 1 centimeter is defined as complete block. The length of filament in centimeters that caused positive response was recorded. in the case of complete block, the value 0 was recorded. As mentioned, after initial corneal sensitivity testing, each eye was tested at 5-min intervals until full sensitivity was restored.

Corneal epithelium defect assessment
in rats administered 0.5% tetracaine that received saline or BPC 157, l-nAMe and/or l-arginine eye drops as described above, corneal epithelium defects considering long-term exposure were assessed at 30, 60 and 150 minutes of the experiment. Corneal epithelium defects were monitored by slit lamp (PSl Portable Slit lamp, reichert, Buffalo, uSA) with blue cobalt filter and by photographing the lesions after staining with standard 10% fluorescein (Alcon, Geneva, Switzerland), according to the experimental protocol. The test was positive if there was an epithelial defect and negative if there was no epithelial defect. Scoring is performed by assessing the area affected with corneal defects divided in quarters, from no defects (0/4) to the whole area of the cornea being affected with epithelium defects (4/4).

Schirmer test assessment
Schirmer test was performed at 30, 60 and 120 min in 0.5% tetracaine rats that received saline or BPC 157, l-nAMe and/or l-arginine eye drops as described above 30 . Standard Schirmer tracks using 2-mm wide test strips cut from Schirmer test paper to fit the size of the rat eye were inserted 1 mm into the inferior conjunctival fornix and placed for 5 minutes, after which results in millimeters were recorded (the values of 5.5±1.0 mm obtained in healthy rats were considered normal).

Statistical analysis
data were processed by use of descriptive statistics according to their distribution (Kolmogorov-Smirnov test for normal distribution). The duration of complete corneal anesthesia (t1) and time interval until corneal sensitivity returned to baseline values (t4) for two different local anesthetic drugs (tetracaine and oxybuprocaine) in regard to different adjuvants (saline, BPC, l-arginine, l-nAMe, and their combinations) were explored by the analysis of variance (AnOVA). differences in the effect of individual adjuvants and their combination on the duration of tetracaine/oxybuprocaine induced corneal anesthesia were analyzed in detail using the Mann-Whitney test for independent samples, followed by Benjamini and hochberg procedure to control false discovery rate (fdr). All statistical analyses were performed using MedCalc 9.5.1.0 (MedCalc Software, Mariakerke, Belgium). The values of p<0.05 were considered statistically significant.

Results
We focused on therapy relation between the stable gastric pentadecapeptide BPC 157, l-nAMe and l-arginine, applied alone and/or in combination, and corneal anesthesia in rats (tables 1-3, figs. 1-4).

Corneal sensitivity measurement
A quite long-lasting corneal insensitivity was induced by 0.5% tetracaine or 0.4% oxybuprocaine in each eye and measured using the Cochet-Bonnet esthesiometer (tables 1-2, figs. 1-2). At 1 minute after two drops of 0.5% tetracaine or 0.4% oxybuprocaine in each eye, rats received locally (two drops/eye) pentadecapeptide BPC 157, l-nAMe, l-arginine, administered alone and in combination. BPC 157 applied as eye drops consistently recovered corneal sensitivity in both tetracaine-and oxybuprocaine-rats. in tetracaine-rats and oxybuprocaine-rats, an indicative point was that the full counteracting effect of the BPC 157 administration also occurred in the presence of nOS-blockade (l-nAMe+BPC 157), as well as in nOS-substrate application (l-arginine+BPC 157). l-arginine eventually shortened the duration of corneal insensitivity. lengthening of corneal insensitivity appeared after l-nAMe. When l-nAMe and larginine were given together (l-nAMe+l-arginine), they antagonized each other's effect (i.e. nOS specific effect), but these rats still exhibited lengthening of corneal insensitivity. Additional BPC 157 co-application (l-nAMe+l-arginine+BPC 157) would have re-established counteraction over topical ophthalmic anesthetic-induced corneal insensitivity.

Corneal epithelium defect assessment
A quite long-lasting 0.5% tetracaine corneal insensitivity was soon associated with development of corneal lesions, sustained consistently until the end of the experiment, which was markedly influenced by pentadecapeptide BPC 157, l-nAMe and l-arginine administered alone and in combination (figs. [3][4]. Much like corneal insensitivity, BPC 157 applied as eye drops consistently reduced development of corneal lesions. likewise, the full counteracting effect of the BPC 157 administration also occurred in the presence of nOS-blockade (l-nAMe+BPC 157), as well as in nOS-substrate application (l-arginine+BPC 157). l-arginine that markedly shortened the duration of corneal insensitivity had only temporary effect on corneal lesions, unable to counteract final lesions. Consistent lengthening of corneal insensitivity with l-nAMe was associated with initial protection reversed to subsequent aggravation. When l-nAMe and larginine were given together (l-nAMe+l-arginine), their mutual antagonizing ensured that these rats, which exhibited lengthening of corneal insensitivity, consistently had fewer lesions. Additional BPC 157 co-application (l-nAMe+l-arginine+BPC 157) resulted consistently in fewer lesions.

Schirmer test assessment
Schirmer test showed that tetracaine application consistently decreased tear volume (table 3). BPC 157 application completely counteracted tetracaine-induced decrease of tear volume and maintained normal healthy values. l-arginine counteracted tetracaine-induced decrease of tear volume only at the earliest interval. l-nAMe initially counteracted but later even augmented tetracaine-induced decrease of tear volume. When given together, l-nAMe+l-arginine, they consistently counteracted tetracaine-induced decrease of tear volume. Additional BPC 157 co-application (l-nAMe+BPC 157, l-arginine+BPC 157, l-nAMe+l-arginine+BPC 157) consistently counteracted tetracaine-induced decrease of tear volume.

Discussion
With the investigated topical ophthalmic anesthetics applied on the surface of the eye and known to act by blocking sodium channels in neuronal axons, thus preventing conduction along the axons and keeping the brain from detecting painful stimuli 26 , BPC 157 applied as eye drops has a particular counteracting Table 3
Thus, we recorded lesion counteraction and eventually less lesion formation in both tetracaine-rats and oxybuprocaine-rats administered BPC 157 therapy. in addition, BPC 157 markedly interferes with atropine effects, reduces atropine-induced mydriasis, and thereby it may increase tear volume once it has been decreased with the application of topical ophthalmic anesthetics on the surface of the eye.
As mentioned above, BPC 157 largely interacts with nOS in different models and species, with both l-nAMe and l-arginine effects [1][2][3][4][5][6][7][8][9][10][11][12][13] . in tetracaine-rats and oxybuprocaine-rats, an indicative point is that the full counteracting effect of BPC 157 administration also occurs in the presence of nOS blockade (l-nAMe+BPC 157) and nOS substrate application (l-arginine+BPC 157). Moreover, it occurs in the circumstances of particular nOS involvement. While both antagonize atropine-induced mydriasis 23 , after topical ophthalmic anesthetics l-nAMe and l-arginine they have a distinctive effect on corneal insensitivity duration, rate of corneal lesions and tear volume. Fig. 3. Corneal epithelium defects, scored 0-4 (Min/Med/Max), course with 0.5% tetracaine corneal anesthesia in rats and effect of pentadecapeptide BPC 157 (0.4 µg/eye), L-NAME (0.1 mg/eye) and/or L-arginine (2 mg/eye) administered alone and in combination after complete corneal anesthesia was established, at 1 min after 0.5% tetracaine corneal anesthesia. After initial corneal sensitivity testing, each eye was tested at 5-minute intervals until full sensitivity was restored; *p<0.05 vs. saline (control).  l-arginine eventually shortens duration of corneal insensitivity, shows counteraction to corneal lesions (and counteraction of tetracaine-induced decrease in tear volume), however, only in the early but not in later period. l-nAMe application was associated with prolonged corneal insensitivity, higher rate of corneal lesions and reduction of tear volume. When l-nAMe and l-arginine were given together (l-nAMe+larginine), they antagonized each other's effect (i.e. nOS specific effect), and these rats exhibited lengthening of corneal insensitivity, but had less corneal lesions. They also counteracted tetracaine-induced decrease of tear volume. Thus, with corneal insensitivity, l-nAMe effect prevailed over l-arginine effect; with less corneal lesions, l-arginine effect prevailed over l-nAMe effect. This distinctive effect (corneal insensitivity, corneal lesion, tear formation) may indicate particular nOS involvement (corneal insensitivity vs. corneal lesion along with tear formation) that may be distinctively affected by the application of nO agents. Anyway, be it in the early or late course, additional BPC 157 co-application (l-nAMe+l-arginine+BPC 157) would have re-established counteraction over topical ophthalmic anesthetic-induced effects (i.e. particular BPC 157 to nOS relations).
Of note, distinctive nOS presentation was also recorded in other studies with various models 23,[31][32][33][34] . This provides a common model 23,[31][32][33][34] that supports particular nOS involvement also beyond the topical ophthalmic anesthetic-induced adverse effects. On the other hand, this combined point observed with BPC 157 administration implicates its particular relations with topical ophthalmic anesthetics and possible counteraction of their adverse effects. unlike topical ophthalmic anesthetic inhibition, with the open eye, there are light-evoked increases in tear volume 35 , thus BPC 157 can permanently (l-arginine only temporarily) evoke protective reflexes such as lacrimation.
The noted counteraction of severe cardiac disturbances (bradycardia, prolongation of all of the observed waves and intervals, AV-block, ventricular ectopy, ventricular tachycardia, t-wave elevation and asystole) after bupivacaine (and counteraction of other various arrhythmias) [14][15][16][17][18][19] appears much like counteraction of adverse effects of tetracaine and oxybuprocaine topical ophthalmic anesthetics applied on the surface of the eye. Greater bupivacaine potential of direct cardiac toxicity than other agents, greater affinity for inactive and resting sodium channel configurations and slower dissociation from these channels 36 together support an even more effective counteraction of all tetracaine-and oxybuprocaine-induced adverse effects, as recorded in our study. Moreover, these findings approach the BPC 157 counteracting effect on the essential chain of the events following local anesthetic application 36,37 . These may be essential for penetration of both the epineurium and neuronal membrane, duration of the action by the time that local anesthetic remains in close proximity to neural fibers, and tetracaine lipid solubility and neuronal protein binding resulting in a local anesthetic that is more potent with an extended duration of action 38 .
Of note, in corneal innervation studies [39][40][41] , mechanical threshold assessed by Belmonte esthesiometer provides structural and functional correlation, and thus corneal nerve function maintenance and recovery. in systemic terms, this may explain why BPC 157 produced analgesia in the MgSO4 and acetic acid intraperitoneal test 29 , and counteracted peritonitis in several models [31][32][33]42 .
likewise, in rats with severed sciatic nerve, BPC 157 markedly improves nerve healing and counteracts autotomy 43 that reflects chronic neuropathic pain, neuroma at the proximal nerve stump, regenerative nerve sprouts growing into all directions, as prevents or, at  least, significantly attenuates the chain of events otherwise leading to the painful sensation referred to as denervated region 44 . A corresponding autotomy counteraction appears in l2-l3 spinal compression model when the stable gastric pentadecapeptide BPC 157 improves the healing course of the spinal cord injury and leads to functional recovery in rats 45 . As mentioned above, much like topical ophthalmic anesthetic-induced adverse effects, BPC 157 counteracts morphine-induced analgesia 46 and counteracts nSAidinduced toxicity 7,47-55 , also known to decrease corneal sensitivity 56 . Therefore, the stable gastric pentadecapeptide BPC 157 can be a quite distinctive 'healing antidote' for adverse effects of topical ophthalmic anesthetics.
Considering the combined vasoconstrictive and local anesthetic properties, BPC 157 may also have a particular effect on vasculature that may influence the duration of topical ophthalmic anesthetic effect. This may be rapid activation of the bypassing loop that occurs in the rat with infrarenal occlusion of the inferior vena cava (and thereby, resolved Virchow), much like in the rats with ischemic/reperfusion colitis, duodenal venous congestion lesions, perforated cecum, bile duct ligation induced liver cirrhosis and portal hypertension [31][32][33]42,57 . Accordingly, BPC 157 interacts with several molecular pathways 1,42,[58][59][60][61][62][63][64] . in particular, BPC 157 increased expression and internalization of VeGfr2, activation of the VeGfr2-Akt-enOS signaling pathway without the need of other known ligands or shear stress 61 .
finally, to counteract topical ophthalmic anesthetic-induced disturbances, it may be that BPC 157 has an effect of its own on membrane potential. inhibition of depolarization of heK293 cells by bupivacaine in vitro fully supports that BPC 157 may, through direct action, successfully counteract the effect of topical ophthalmic anesthetics, much like the effect of bupivacaine 14 .
likewise, cell depolarization due to increasing magnesium concentration was inhibited in the presence of BPC 157 (1 µM) in vitro 34 . furthermore, BPC 157 reduced depolarization caused by hyperkalemia in heK293 cells 15 . depolarization caused by BPC 157 (1 µm) was inhibited by the application of the nonspecific potassium blocker BaCl2 (1 mM) 15 . in conclusion, we suggest BPC 157 as an antidote of topical ophthalmic anesthetics.