Int Ophthalmol DOI 10.1007/s10792-016-0184-9
ORIGINAL PAPER
Effect of topical administration of tramadol on corneal wound healing in rats Ozgun Cuvas Apan . Murat Atabey Ozer . Selcuk Takir . Alparslan Apan . Demet Sengul
Received: 4 November 2015 / Accepted: 19 January 2016 Ó Springer Science+Business Media Dordrecht 2016
Abstract In this study, we aimed to investigate the effects of topical tramadol administration on corneal wound healing, and examine ophthalmic structures and intraocular pressure 7 days after tramadol administration. The experiments were conducted on eight male Wistar rats (250–300 g). After ophthalmic examination, epithelial cell layers in the central cornea were wounded. Rats received 30 lL of tramadol hydrochloride in one eye (Group Tramadol) and the same volume of vehicle in the other (Group Control) every 12 h for 7 days. Both eyes were stained with fluorescein dye, photographed, and wound area was calculated every 8 h until complete healing was observed. Eye blink frequency and corneal reflex tests were measured before and after drug administrations. After 7 days, slit lamp O. Cuvas Apan (&) A. Apan Department of Anesthesiology and Intensive Care Medicine, Giresun University Medical Faculty, Nizamiye Yerleskesi, Orhan Yılmaz Caddesi, Mumcular Sokak No:1, Merkez, 28200 Giresun, Turkey e-mail: [email protected] M. A. Ozer Department of Ophthalmology, Giresun University Medical Faculty, Giresun, Turkey S. Takir Department of Clinical Pharmacology, Giresun University Medical Faculty, Giresun, Turkey D. Sengul Department of Pathology, Giresun University Medical Faculty, Giresun, Turkey
biomicroscopy, fundoscopy, Goldmann applanation tonometry, and histological evaluation were performed. There was no difference in the corneal wound healing rates between the tramadol and control groups. Reduction in wound area over time was also similar; grouptime interaction was insignificant (F = 738.911; p = 0.225). Tramadol application resulted in blinking and blepharospasm for 30 s, but vehicle did not. Corneal reflex was intact and eye blink frequency test results were similar in all measurement times in both groups. Slit lamp biomicroscopy, fundoscopy, and intraocular pressures were within normal range. Corneal cells appeared unaffected by the repeated doses of tramadol for 7 days. Topical tramadol application on the cornea did not cause any side effect, except for initial temporary blinking and blepharospasm. Corneal wound healing was not affected, either. Keywords Tramadol Administration Topical Cornea Wound healing
Introduction Tramadol is an effective analgesic widely used for the relief of acute and chronic pain. It is a racemic mixture of two enantiomers, namely (?) tramadol and (-) tramadol. Tramadol has opiate activity on l, j, and d receptors; causes a-2 adrenergic stimulation; inhibits norepinephrine and serotonin reuptake; and enhances serotonin release [1, 2]. Local analgesic effect of
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tramadol has been shown to be similar to local anesthetics [3–5]. Tramadol also acts as a TRPV1 (transient receptor potential channel, subfamily V, member 1) agonist. Activation of TRPV1 on sensory neurons is followed by a local release of vasoactive neuropeptides and a marked desensitization of the afferent fibers, hence termination of pain sensation [6]. The cornea is primarily innervated by small-diameter C-fiber sensory neurons expressing TRPV1, a sodium/calcium cation channel expressed abundantly by nociceptive neurons, and consequently a target for pain control [7]. Long-lasting pain relief without adverse effects following corneal abrasions or corneal ulcers is difficult to achieve with existing analgesics. In this study, we aimed to investigate the effects of topical tramadol administration on corneal wound healing. We also intended to examine ophthalmic structures and intraocular pressure (IOP) 7 days after topical tramadol administration. This is, to our knowledge, the first study to evaluate the effects of topical tramadol administration on corneal wound healing in rats with experimental corneal wounds.
Methods All experiments were carried out according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and recommendations of the IASP [8], and were approved by the Giresun University Animal Experiments Local Ethics Committee (Giresun, Turkey). Experiments were conducted on eight male Wistar rats (250–300 g) obtained from a laboratory animals farm (Saki Yenili Laboratory Animals Farm, Ankara, Turkey). They were housed under a 12 h alternating light–dark cycle, and had access to food and water ad libitum. All rats underwent ophthalmic examination including direct ophthalmoscopy. The rats (n = 8) were anesthetized with ketamine (50 mg/kg) plus xylazine (5 mg/kg) through intra-peritoneal (IP) route. After abolition of the reflex responses to touch stimuli to tail, hind leg and eyelid, and body movement cessation, slit lamp biomicroscopy, fundoscopy, and Goldmann applanation tonometry tests were performed. An ophthalmic caliper (Janach J2820.4 Janach Inc., Italy) was placed in the center of the cornea, and a circle with a diameter of 3 mm was marked under biomicroscopy. Epithelial cell layers in the marked area were debrided mechanically. Both eyes were stained with
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fluorescein dye and photographed, then total corneal area and corneal wound area were calculated. Following recovery from anesthesia, eye blink frequency was measured for 1 min while rats were in their resting position. Corneal reflex was tested by touching a sterile cotton swab to the lateral corner of the cornea. Enrofloxacin (Baytril 2.5 % oral solution) 10 mg/kg was administered twice daily by oral gavage for 3 days to prevent infection. Rats received 30 lL (5.2 mg/kg) of tramadol hydrochloride (Tramosel 100 mg/2 ml ampoule, Haver Drug Industry, Istanbul, Turkey) in one eye (Group Tramadol) and the same volume of vehicle in the other (Group Control). An artificial tear (hydroxypropyl methylcellulose) was used as vehicle (Tears NaturaleÒ Free eye drop 0.8 ml flacon, Alcon, United States). Ten microliters were pipetted directly onto the corneal surface, and this application was repeated three times consecutively to reach the required volume, in both eyes. Reactions to the application such as wiping, blinking, or blepharospasm (incomplete blinks) were observed. Topical applications of the drug and vehicle were performed every 12 h for 7 days. Eye blink frequency and corneal reflex tests were measured before and 5, 15, 30, 60, and 90 min after drug administrations until corneal healing were completed. Both eyes were stained with fluorescein dye and photographed every 8 h until complete healing was observed. Measurements were done from enlarged photographs of the wounds. Total corneal area and corneal wound area were calculated using pixel count method (Photoshop CS6) (Fig. 1). After drug application for 7 days, the rats were anesthetized with ketamine (50 mg/kg) plus xylazine (5 mg/kg) through intraperitoneal (IP) route. Slit lamp biomicroscopy, fundoscopy, and Goldmann applanation tonometry tests were performed. Then rats were euthanized by intraperitoneal injection of an overdose of sodium thiopental. Control and treated eyes were histologically compared. Eyes were enucleated, fixed in 10 % buffered formaldehyde solution for 48 h, embedded in paraffin, and sectioned at 5 microns. Tissue was stained with hematoxylin-eosin (H&E) and evaluated under a light microscope (10 9 0.25). Statistical analysis Data were expressed as median (25th–75th percentile), mean ± SD or percentage (%). Variables in the tramadol group were compared with those in the
Int Ophthalmol
Fig. 1 Corneal wound area was calculated using pixel count method. Total corneal area (a), corneal wound area (b)
control group with the Mann–Whitney U test. Within each group, comparisons of variables before versus after treatment and the differences between baseline values and the values measured at each subsequent time point were performed by the Wilcoxon signedrank test. Assessment of significance in average wound area in tramadol- and vehicle-treated eyes over time was made repeated measures of ANOVA. Statistical analysis was performed using Statistical Package for the Social Sciences (SPSS) 11.5 (SPSS Inc., Chicago, IL, United States). Differences were considered significant at a value of p \ 0.05.
Results There was no difference in the corneal wound healing rates between the tramadol and control groups (Fig. 2). Complete healing was observed at 40 h post-debridement (Fig. 3). Reduction in wound area over time was also similar, in other words group-time interaction was insignificant (F = 738.911; p = 0.225). Topical application of the drug or vehicle caused wiping movement in all rats. Tramadol application also resulted in blinking and blepharospasm for 30 s, but vehicle did not. Corneal reflex was intact and eye blink frequency test results were similar in all measurement times in both groups (Fig. 4). After we observed initial blinking and short term blepharospasm response to tramadol, we dropped a local anesthetic (30 ll of lidocaine hydrochloride 2 %) 5 min before tramadol administration in another two healthy rats not included in the study group. Lidocaine pretreatment completely eliminated tramadol induced eye blinking and blepharospasm. Slit lamp biomicroscopy, fundoscopy, and Goldmann applanation tonometry tests were within normal
Fig. 2 Reduction in wound area (%) over time in two groups. Group T tramadol, Group C control
range before debridement. There was no significant difference in these tests’ results 7 days after drug application. Baseline IOP was 14.5 (13.3–16.0) mmHg in group T, and 14 (13.0–15.8) mmHg in Group C (p = 0.670). Intracular pressure 7 days after drug application was 15.5 (13.5–16.8) mmHg in group T, and 14.0 (14.0–15.8) mmHg in Group C (p = 0.786). There was no statistically significant difference between baseline and second IOP measurements within each group (p = 0.785 in Group T, and p = 0.703 in Group C, respectively). Histomorphological features of the cornea (Fig. 5), lens, retina, and optic nerve were also normal in each group.
Discussion This study demonstrates that topical administration of tramadol did not impair corneal wound healing, nor did it affect corneal reflex and eye blink frequency. It
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Fig. 3 Corneas were stained with fluorescein dye and photographed at 0 (a), 8, 16 (b), 24, 32, and 40 h (c). Complete reepithelialization occurred by 40 h in all eyes (c)
Fig. 4 Eye blink frequency before (0) and 5, 15, 30, 60, and 90 min after drug administration in the first (a) and second (b) days. T tramadol, C control, 1 first application, 2 second application
Fig. 5 Tramadol-treated (a) and vehicle-treated (b) corneas were histologically indistinguishable using H&E staining (10 9 0.25)
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caused no apparent damage to the cornea 7 days after its administration. Tramadol did not affect intraocular pressure, either. There are no data in the literature related to topical tramadol application on the cornea. One interesting case report is available about peribulbar administration of tramadol, clonidine, and ropivacaine mixture in blind and intact painful eyes in two elderly patients. The researchers reported that sustained relief of ocular pain was achieved, and no side effect was observed [9]. On the other hand, all of these effects cannot be attributed only to tramadol. Topical administration, mostly in the form of eye drops, is noninvasive, self-administrable, and has a high patient compliance. Topical local anesthetics are precluded from chronic use (i.e., several days of analgesia) due to direct corneal toxicity [10] and are, therefore, not appropriate for post-operative pain control during recovery from ophthalmic surgery. Topical nonsteroidal anti-inflammatory drugs (NSAIDs) have toxic effects on corneal epithelial cells [11], and systemic NSAIDs can produce side effects, such as gastric ulceration and renal toxicity [12]. Topical use of morphine relieves pain associated with corneal wounds in dogs [13], and rats [14]. Additionally, in a report, morphine did not lead to a delay in corneal wound healing when it was used topically [13]; but unfortunately, it has an abuse potential. Tramadol has not been associated with clinically significant respiratory depression [15], and has a low potential for the development of tolerance, dependence, and abuse [16]. The corneal epithelium has several cell layers and appears to present a significant barrier to penetration of the drug. After topical drug application, only a thin film makes direct contact with the cornea and there is likely a local dilution effect due to tear production [17]. Therefore, we applied a higher dose than used in systemic administration in clinical practice, and total dose was administered in repeated small volumes to reduce waste. The dose was consistent with the previous study about pharmacokinetics of tramadol in rats [18]. The corneal epithelium is lipoidal in nature which contains 90 % of the total cells in the cornea and poses a significant resistance for permeation of topically administered hydrophilic drugs. It is vital to understand that the drug should have an amphipathic nature in order to permeate through corneal layers [17] Only moderately charged small molecules are able to
penetrate through the cornea [19, 20]. Tramadol’s high solubility and lipophilicity may offer advantage for topical application to cornea. It has a molecular weight of 299.8. It is very soluble in water; is freely soluble in ethanol; and has a pKa of 9.41 [18]. Tramadol achieves its analgesic activity not only by affecting opioidergic receptors and noradrenalin, serotonin reuptake inhibition [1, 2], but also by showing an agonistic effect on TRPV1 [6]. TRPV1 of the corneal neurons works in close relation to substance P (SP) and calcitonin-gene-related peptide (CGRP) both in the cornea and central nerve system for healing and nociceptive transduction [21]. In an experimental study, investigators showed that topical administration of resiniferatoxin (RTX), a potent TRPV1 agonist, on cornea has provided prolonged analgesia. The authors stated that RTX has not impaired corneal wound healing [7]. In our study, tramadol application on cornea resulted in blinking and blepharospasm for 30 s. Topical application of lidocaine hydrochloride completely eliminated tramadol induced eye blinking and blepharospasm observed for 30 s. This response supports agonistic activity of tramadol on TRPV1, because TRPV1 activity is responsible for local side effects (e.g., initiation of burning pain) of tramadol [6]. We continued topical tramadol administration for 7 days. In painful situations, it is required to maintain the analgesic treatment after corneal healing. In addition, treatment may be prolonged in some cases. Some stromal ulcers may be classified as nonprogressive and treated similarly to superficial ulcerations. Others are observed to be progressive as indicated by increasing depth and width of the ulcer, and they need more aggressive and long-lasting treatment [22, 23]. Medications preventing corneal healing such as NSAIDs or local anesthetics may cause corneal perforation. Tramadol may be beneficial for pain relief in corneal abrasions, superficial ulcers, and deep stromal ulcers related to trauma, burn, and disorders due to inflammation. When we take into account the side effects of the chronic local anesthetic or NSAIDs, an analgesic having fewer ocular and systemic side effects is worth investigating. When assessed using fluorescein dye and histology, corneal cells appeared unaffected by the repeated doses of tramadol for 7 days. Intraocular pressure was not affected. Tramadol also preserved protection mechanisms such as corneal reflex and eye blink, unlike local anesthetics. Although tramadol also has local anesthetic activity
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[3–5], the dose and administration route of the drug used in the study did not result in reduced mechanosensitive responses. Cellular expression of TRPV1 leaves non-nociceptive neurons and mechanosensitive nociceptive neurons functionally intact [24]. One limitation of this study is that we assessed mechanosensitive responses, and according to our results, it is impossible to evaluate tramadol’s analgesic effect on the cornea by this method. Sample size is also a limitation. Another limitation is that tramadol has a complex mechanism on analgesia; therefore, the results of this study cannot be attributed to a certain mechanism. In conclusion, topical tramadol application on the cornea did not cause any side effect, except initial temporary blinking and blepharospasm. Corneal wound healing was not affected, either. Tramadol in the form of eye drop should be investigated as an alternative to local anesthetics, opioids, or NSAIDs for the treatment of ocular pain.
7.
8.
9.
10. 11.
12.
13.
14. Funding
This research was carried out without funding. 15.
Compliance with ethical standards Conflict of interest The authors declare that they have no conflict of interest. Ethical approval All applicable international, national, and/ or institutional guidelines for the care and use of animals were followed.
References 1. Eggers KA, Power I (1995) Tramadol. Br J Anaesth 74:247–248 2. Lewis KS, Han NH (1997) Tramadol: a new centrally acting analgesic. Am J Health-Syst Pharm 54:643–652 3. Bianchi M, Rossoni G, Sacerdote P, Panerai AE (1999) Effects of tramadol on experimental inflammation. Fundam Clin Pharmacol 13:220–225 4. Tsai YC, Chang PJ, Jou IM (2001) Direct tramadol application on sciatic nerve inhibits spinal somatosensory evoked potentials in rats. Anesth Analg 92:1547–1551 5. Sousa AM, Ashmawi HA, Costa LS, Posso IP, Slullitel A (2012) Percutaneous sciatic nerve block with tramadol induces analgesia and motor blockade in two animal pain models. Braz J Med Biol Res 45:147–152 6. Marincsa´k R, To´th BI, Czifra G, Szabo T, Kovacs L, Biro T (2008) The analgesic drug, tramadol. Acts as an agonist of
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the transient receptor potentialvanilloid-1. Anesth Analg 106:1890–1896 Bates B, Mitchell K, Keller JM, Chan CC, Swaim WD, Yaskovich R, Andrew J, Mannes AJ, Iadarola MJ (2010) Prolonged analgesic response of cornea to topical resiniferatoxin, a potent TRPV1 agonist. Pain 149:522–528 Zimmermann M (1983) Ethical guidelines for investigations of experimental pain in conscious animals. Pain 16:109–110 Tarantini A, Lanzetta P, Cugini U (2007) Peribulbar tramadol, clonidine, and ropivacaine in blind and seeing painful eyes. Eur J Ophthalmol 17:976–978 McGee HT, Fraunfelder FW (2007) Toxicities of topical ophthalmic anesthetics. Expert Opin Drug Saf 6:637–640 Hendrix DV, Ward DA, Barnhill MA (2002) Effects of antiinflammatory drugs and preservatives on morphologic characteristics and migration of canine corneal epithelial cells in tissue culture. Vet Ophthalmol 5:127–135 Giuliano EA (2004) Nonsteroidal anti-inflammatory drugs in veterinary ophthalmology. Vet Clin North Am Small Anim Pract 34:707–723 Stiles J, Honda CN, Krohne SG, Kazacos EA (2003) Effect of topical administration of 1% morphine sulfate solution on signs of pain and corneal wound healing in dogs. Am J Vet Res 64:813–818 Wenk HN, Nannenga MN, Honda CN (2003) Effect of morphine sulphate eye drops on hyperalgesia in the rat cornea. Pain 105:455–465 Vickers MD, O’Flaherty D, Szekely SM, Read M, Yoshizumi J (1992) Tramadol: pain relief by an opioid without depression of respiration. Anaesthesia 47:291–296 Preston KL, Jasinski DR, Testa M (1991) Abuse potential and pharmacological comparison of tramadol and morphine. Drug Alcohol Depend 27:7–17 Gaudana P, Ananthula HK, Parenky A, Mitra AK (2010) Ocular drug delivery. AAPS J 12:348–360 Zhao Y, Tao T, Wu J, Pi J, He N, Chai X, Chen Q (2008) Pharmacokinetics of tramadol in rat plasma and cerebrospinal fluid after intranasal administration. J Pharm Pharmacol 60:1149–1154 Edwards A, Prausnitz MR (2001) Predicted permeability of the cornea to topical drugs. Pharm Res 18:1497–1508 Prausnitz MR, Noonan JS (1998) Permeability of cornea, sclera, and conjunctiva: a literature analysis for drug delivery to the eye. J Pharm Sci 87:1479–1488 Murata Y, Masuko S (2006) Peripheral and central distribution of TRPV1, substance P and CGRP of rat corneal neurons. Brain Res 1085:87–94 Wipperman JL, Dorsch JN (2013) Evaluation and management of corneal abrasions. Am Fam Physician 87:114–120 Belknap EB (2015) Corneal emergencies. Topics Companion Anim Med 30:74–80 Karai L, Brown DC, Mannes AJ, Connelly ST, Brown J, Gandal M, Wellisch OM, Neubert JK, Olah Z, Iadarola MJ (2004) Deletion of vanilloid receptor 1-expressing primary afferent neurons for pain control. J Clin Invest 113:1344–1352
ORIGINAL PAPER
Effect of topical administration of tramadol on corneal wound healing in rats Ozgun Cuvas Apan . Murat Atabey Ozer . Selcuk Takir . Alparslan Apan . Demet Sengul
Received: 4 November 2015 / Accepted: 19 January 2016 Ó Springer Science+Business Media Dordrecht 2016
Abstract In this study, we aimed to investigate the effects of topical tramadol administration on corneal wound healing, and examine ophthalmic structures and intraocular pressure 7 days after tramadol administration. The experiments were conducted on eight male Wistar rats (250–300 g). After ophthalmic examination, epithelial cell layers in the central cornea were wounded. Rats received 30 lL of tramadol hydrochloride in one eye (Group Tramadol) and the same volume of vehicle in the other (Group Control) every 12 h for 7 days. Both eyes were stained with fluorescein dye, photographed, and wound area was calculated every 8 h until complete healing was observed. Eye blink frequency and corneal reflex tests were measured before and after drug administrations. After 7 days, slit lamp O. Cuvas Apan (&) A. Apan Department of Anesthesiology and Intensive Care Medicine, Giresun University Medical Faculty, Nizamiye Yerleskesi, Orhan Yılmaz Caddesi, Mumcular Sokak No:1, Merkez, 28200 Giresun, Turkey e-mail: [email protected] M. A. Ozer Department of Ophthalmology, Giresun University Medical Faculty, Giresun, Turkey S. Takir Department of Clinical Pharmacology, Giresun University Medical Faculty, Giresun, Turkey D. Sengul Department of Pathology, Giresun University Medical Faculty, Giresun, Turkey
biomicroscopy, fundoscopy, Goldmann applanation tonometry, and histological evaluation were performed. There was no difference in the corneal wound healing rates between the tramadol and control groups. Reduction in wound area over time was also similar; grouptime interaction was insignificant (F = 738.911; p = 0.225). Tramadol application resulted in blinking and blepharospasm for 30 s, but vehicle did not. Corneal reflex was intact and eye blink frequency test results were similar in all measurement times in both groups. Slit lamp biomicroscopy, fundoscopy, and intraocular pressures were within normal range. Corneal cells appeared unaffected by the repeated doses of tramadol for 7 days. Topical tramadol application on the cornea did not cause any side effect, except for initial temporary blinking and blepharospasm. Corneal wound healing was not affected, either. Keywords Tramadol Administration Topical Cornea Wound healing
Introduction Tramadol is an effective analgesic widely used for the relief of acute and chronic pain. It is a racemic mixture of two enantiomers, namely (?) tramadol and (-) tramadol. Tramadol has opiate activity on l, j, and d receptors; causes a-2 adrenergic stimulation; inhibits norepinephrine and serotonin reuptake; and enhances serotonin release [1, 2]. Local analgesic effect of
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tramadol has been shown to be similar to local anesthetics [3–5]. Tramadol also acts as a TRPV1 (transient receptor potential channel, subfamily V, member 1) agonist. Activation of TRPV1 on sensory neurons is followed by a local release of vasoactive neuropeptides and a marked desensitization of the afferent fibers, hence termination of pain sensation [6]. The cornea is primarily innervated by small-diameter C-fiber sensory neurons expressing TRPV1, a sodium/calcium cation channel expressed abundantly by nociceptive neurons, and consequently a target for pain control [7]. Long-lasting pain relief without adverse effects following corneal abrasions or corneal ulcers is difficult to achieve with existing analgesics. In this study, we aimed to investigate the effects of topical tramadol administration on corneal wound healing. We also intended to examine ophthalmic structures and intraocular pressure (IOP) 7 days after topical tramadol administration. This is, to our knowledge, the first study to evaluate the effects of topical tramadol administration on corneal wound healing in rats with experimental corneal wounds.
Methods All experiments were carried out according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and recommendations of the IASP [8], and were approved by the Giresun University Animal Experiments Local Ethics Committee (Giresun, Turkey). Experiments were conducted on eight male Wistar rats (250–300 g) obtained from a laboratory animals farm (Saki Yenili Laboratory Animals Farm, Ankara, Turkey). They were housed under a 12 h alternating light–dark cycle, and had access to food and water ad libitum. All rats underwent ophthalmic examination including direct ophthalmoscopy. The rats (n = 8) were anesthetized with ketamine (50 mg/kg) plus xylazine (5 mg/kg) through intra-peritoneal (IP) route. After abolition of the reflex responses to touch stimuli to tail, hind leg and eyelid, and body movement cessation, slit lamp biomicroscopy, fundoscopy, and Goldmann applanation tonometry tests were performed. An ophthalmic caliper (Janach J2820.4 Janach Inc., Italy) was placed in the center of the cornea, and a circle with a diameter of 3 mm was marked under biomicroscopy. Epithelial cell layers in the marked area were debrided mechanically. Both eyes were stained with
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fluorescein dye and photographed, then total corneal area and corneal wound area were calculated. Following recovery from anesthesia, eye blink frequency was measured for 1 min while rats were in their resting position. Corneal reflex was tested by touching a sterile cotton swab to the lateral corner of the cornea. Enrofloxacin (Baytril 2.5 % oral solution) 10 mg/kg was administered twice daily by oral gavage for 3 days to prevent infection. Rats received 30 lL (5.2 mg/kg) of tramadol hydrochloride (Tramosel 100 mg/2 ml ampoule, Haver Drug Industry, Istanbul, Turkey) in one eye (Group Tramadol) and the same volume of vehicle in the other (Group Control). An artificial tear (hydroxypropyl methylcellulose) was used as vehicle (Tears NaturaleÒ Free eye drop 0.8 ml flacon, Alcon, United States). Ten microliters were pipetted directly onto the corneal surface, and this application was repeated three times consecutively to reach the required volume, in both eyes. Reactions to the application such as wiping, blinking, or blepharospasm (incomplete blinks) were observed. Topical applications of the drug and vehicle were performed every 12 h for 7 days. Eye blink frequency and corneal reflex tests were measured before and 5, 15, 30, 60, and 90 min after drug administrations until corneal healing were completed. Both eyes were stained with fluorescein dye and photographed every 8 h until complete healing was observed. Measurements were done from enlarged photographs of the wounds. Total corneal area and corneal wound area were calculated using pixel count method (Photoshop CS6) (Fig. 1). After drug application for 7 days, the rats were anesthetized with ketamine (50 mg/kg) plus xylazine (5 mg/kg) through intraperitoneal (IP) route. Slit lamp biomicroscopy, fundoscopy, and Goldmann applanation tonometry tests were performed. Then rats were euthanized by intraperitoneal injection of an overdose of sodium thiopental. Control and treated eyes were histologically compared. Eyes were enucleated, fixed in 10 % buffered formaldehyde solution for 48 h, embedded in paraffin, and sectioned at 5 microns. Tissue was stained with hematoxylin-eosin (H&E) and evaluated under a light microscope (10 9 0.25). Statistical analysis Data were expressed as median (25th–75th percentile), mean ± SD or percentage (%). Variables in the tramadol group were compared with those in the
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Fig. 1 Corneal wound area was calculated using pixel count method. Total corneal area (a), corneal wound area (b)
control group with the Mann–Whitney U test. Within each group, comparisons of variables before versus after treatment and the differences between baseline values and the values measured at each subsequent time point were performed by the Wilcoxon signedrank test. Assessment of significance in average wound area in tramadol- and vehicle-treated eyes over time was made repeated measures of ANOVA. Statistical analysis was performed using Statistical Package for the Social Sciences (SPSS) 11.5 (SPSS Inc., Chicago, IL, United States). Differences were considered significant at a value of p \ 0.05.
Results There was no difference in the corneal wound healing rates between the tramadol and control groups (Fig. 2). Complete healing was observed at 40 h post-debridement (Fig. 3). Reduction in wound area over time was also similar, in other words group-time interaction was insignificant (F = 738.911; p = 0.225). Topical application of the drug or vehicle caused wiping movement in all rats. Tramadol application also resulted in blinking and blepharospasm for 30 s, but vehicle did not. Corneal reflex was intact and eye blink frequency test results were similar in all measurement times in both groups (Fig. 4). After we observed initial blinking and short term blepharospasm response to tramadol, we dropped a local anesthetic (30 ll of lidocaine hydrochloride 2 %) 5 min before tramadol administration in another two healthy rats not included in the study group. Lidocaine pretreatment completely eliminated tramadol induced eye blinking and blepharospasm. Slit lamp biomicroscopy, fundoscopy, and Goldmann applanation tonometry tests were within normal
Fig. 2 Reduction in wound area (%) over time in two groups. Group T tramadol, Group C control
range before debridement. There was no significant difference in these tests’ results 7 days after drug application. Baseline IOP was 14.5 (13.3–16.0) mmHg in group T, and 14 (13.0–15.8) mmHg in Group C (p = 0.670). Intracular pressure 7 days after drug application was 15.5 (13.5–16.8) mmHg in group T, and 14.0 (14.0–15.8) mmHg in Group C (p = 0.786). There was no statistically significant difference between baseline and second IOP measurements within each group (p = 0.785 in Group T, and p = 0.703 in Group C, respectively). Histomorphological features of the cornea (Fig. 5), lens, retina, and optic nerve were also normal in each group.
Discussion This study demonstrates that topical administration of tramadol did not impair corneal wound healing, nor did it affect corneal reflex and eye blink frequency. It
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Fig. 3 Corneas were stained with fluorescein dye and photographed at 0 (a), 8, 16 (b), 24, 32, and 40 h (c). Complete reepithelialization occurred by 40 h in all eyes (c)
Fig. 4 Eye blink frequency before (0) and 5, 15, 30, 60, and 90 min after drug administration in the first (a) and second (b) days. T tramadol, C control, 1 first application, 2 second application
Fig. 5 Tramadol-treated (a) and vehicle-treated (b) corneas were histologically indistinguishable using H&E staining (10 9 0.25)
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caused no apparent damage to the cornea 7 days after its administration. Tramadol did not affect intraocular pressure, either. There are no data in the literature related to topical tramadol application on the cornea. One interesting case report is available about peribulbar administration of tramadol, clonidine, and ropivacaine mixture in blind and intact painful eyes in two elderly patients. The researchers reported that sustained relief of ocular pain was achieved, and no side effect was observed [9]. On the other hand, all of these effects cannot be attributed only to tramadol. Topical administration, mostly in the form of eye drops, is noninvasive, self-administrable, and has a high patient compliance. Topical local anesthetics are precluded from chronic use (i.e., several days of analgesia) due to direct corneal toxicity [10] and are, therefore, not appropriate for post-operative pain control during recovery from ophthalmic surgery. Topical nonsteroidal anti-inflammatory drugs (NSAIDs) have toxic effects on corneal epithelial cells [11], and systemic NSAIDs can produce side effects, such as gastric ulceration and renal toxicity [12]. Topical use of morphine relieves pain associated with corneal wounds in dogs [13], and rats [14]. Additionally, in a report, morphine did not lead to a delay in corneal wound healing when it was used topically [13]; but unfortunately, it has an abuse potential. Tramadol has not been associated with clinically significant respiratory depression [15], and has a low potential for the development of tolerance, dependence, and abuse [16]. The corneal epithelium has several cell layers and appears to present a significant barrier to penetration of the drug. After topical drug application, only a thin film makes direct contact with the cornea and there is likely a local dilution effect due to tear production [17]. Therefore, we applied a higher dose than used in systemic administration in clinical practice, and total dose was administered in repeated small volumes to reduce waste. The dose was consistent with the previous study about pharmacokinetics of tramadol in rats [18]. The corneal epithelium is lipoidal in nature which contains 90 % of the total cells in the cornea and poses a significant resistance for permeation of topically administered hydrophilic drugs. It is vital to understand that the drug should have an amphipathic nature in order to permeate through corneal layers [17] Only moderately charged small molecules are able to
penetrate through the cornea [19, 20]. Tramadol’s high solubility and lipophilicity may offer advantage for topical application to cornea. It has a molecular weight of 299.8. It is very soluble in water; is freely soluble in ethanol; and has a pKa of 9.41 [18]. Tramadol achieves its analgesic activity not only by affecting opioidergic receptors and noradrenalin, serotonin reuptake inhibition [1, 2], but also by showing an agonistic effect on TRPV1 [6]. TRPV1 of the corneal neurons works in close relation to substance P (SP) and calcitonin-gene-related peptide (CGRP) both in the cornea and central nerve system for healing and nociceptive transduction [21]. In an experimental study, investigators showed that topical administration of resiniferatoxin (RTX), a potent TRPV1 agonist, on cornea has provided prolonged analgesia. The authors stated that RTX has not impaired corneal wound healing [7]. In our study, tramadol application on cornea resulted in blinking and blepharospasm for 30 s. Topical application of lidocaine hydrochloride completely eliminated tramadol induced eye blinking and blepharospasm observed for 30 s. This response supports agonistic activity of tramadol on TRPV1, because TRPV1 activity is responsible for local side effects (e.g., initiation of burning pain) of tramadol [6]. We continued topical tramadol administration for 7 days. In painful situations, it is required to maintain the analgesic treatment after corneal healing. In addition, treatment may be prolonged in some cases. Some stromal ulcers may be classified as nonprogressive and treated similarly to superficial ulcerations. Others are observed to be progressive as indicated by increasing depth and width of the ulcer, and they need more aggressive and long-lasting treatment [22, 23]. Medications preventing corneal healing such as NSAIDs or local anesthetics may cause corneal perforation. Tramadol may be beneficial for pain relief in corneal abrasions, superficial ulcers, and deep stromal ulcers related to trauma, burn, and disorders due to inflammation. When we take into account the side effects of the chronic local anesthetic or NSAIDs, an analgesic having fewer ocular and systemic side effects is worth investigating. When assessed using fluorescein dye and histology, corneal cells appeared unaffected by the repeated doses of tramadol for 7 days. Intraocular pressure was not affected. Tramadol also preserved protection mechanisms such as corneal reflex and eye blink, unlike local anesthetics. Although tramadol also has local anesthetic activity
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[3–5], the dose and administration route of the drug used in the study did not result in reduced mechanosensitive responses. Cellular expression of TRPV1 leaves non-nociceptive neurons and mechanosensitive nociceptive neurons functionally intact [24]. One limitation of this study is that we assessed mechanosensitive responses, and according to our results, it is impossible to evaluate tramadol’s analgesic effect on the cornea by this method. Sample size is also a limitation. Another limitation is that tramadol has a complex mechanism on analgesia; therefore, the results of this study cannot be attributed to a certain mechanism. In conclusion, topical tramadol application on the cornea did not cause any side effect, except initial temporary blinking and blepharospasm. Corneal wound healing was not affected, either. Tramadol in the form of eye drop should be investigated as an alternative to local anesthetics, opioids, or NSAIDs for the treatment of ocular pain.
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14. Funding
This research was carried out without funding. 15.
Compliance with ethical standards Conflict of interest The authors declare that they have no conflict of interest. Ethical approval All applicable international, national, and/ or institutional guidelines for the care and use of animals were followed.
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