Previously published procedures for pONT were modified and performed in animals anesthetized with isoflurane gas and topical proparacaine 1% eye drops (11 (link)). An incision was made in the temporal conjunctiva rather than the superior conjunctiva due to easier access to the nerve behind the globe without excessive retraction of the globe. A diamond knife for radial keratotomy was used to incise the optic nerve to a depth of one third of its diameter 2–3 mm behind the eye. A metal guard limited penetration and variation of incision distance. Prior to the procedure, the diameter of control optic nerves of adult Wistar rats (n=20) from histological optic nerve cross sections previously collected was estimated. The averaged diameter was found to be approximately 0.87 mm and, therefore, the depth of the blade was adjusted to 0.28 mm. A partial cross section of the optic nerve was made carefully, so as not to damage the adjacent blood supply. All eyes were examined ophthalmoscopically to ensure complete retinal blood flow. The conjunctival incision was sutured and topical ophthalmic ointment (tobramycin, Tobrex; Alcon, Fort Worth, TX, USA) was applied immediately after the surgical procedures and then twice daily for 2 days. Surgical procedures were performed on one eye of each rat, the contralateral eye serving as an untreated control.
Tobrex
Manufactured by Alcon
Sourced in Spain, United States, Belgium, Hungary
Tobrex is a topical ophthalmic product manufactured by Alcon. It contains the active ingredient tobramycin, an antibiotic used to treat bacterial infections in the eye.
Automatically generated - may contain errors
Lab products found in correlation
Tobramycin, by Alcon (5 mentions)
Domitor, by Pfizer (4 mentions)
Antisedan, by Pfizer (4 mentions)
Ketolar, by Pfizer (3 mentions)
Romp n, by Bayer (2 mentions)
Rompum, by Bayer (2 mentions)
C57bl 6j wild type, by Charles River Laboratories (1 mentions)
Invivovue diver 2, by Bioptigen (1 mentions)
Envisu r2210, by Bioptigen (1 mentions)
Rompun, by Bayer (1 mentions)
Medetomidine, by Virbac (1 mentions)
Medetor, by Virbac (1 mentions)
Isoflurane, by Abbott (1 mentions)
1 m naoh, by Avantor (1 mentions)
4 0 silk suture, by Johnson & Johnson (1 mentions)
Metacam, by Boehringer Ingelheim (1 mentions)
Balanced salt solution, by Alcon (1 mentions)
Meloxicam, by Boehringer Ingelheim (1 mentions)
Fluospheres polystyrene microspheres, by Thermo Fisher Scientific (1 mentions)
Dumont 7 cross action forceps, by Fine Science Tools (1 mentions)
23 protocols using tobrex
1
Partial Optic Nerve Transection in Rats
2
Murine Model of Ocular Interventions
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All experiments were performed using 8–12 weeks old C57Bl/6J wild-type (WT, in total 20 animals obtained from Charles River Laboratories) mice of both sexes. All animals were housed under standard laboratory conditions according to a normal day/night rhythm with ad libitum access to food and water. For anaesthesia, a mixture of xylazine (10 mg/kg body weight) and ketamine (70 mg/kg body weight, Anesketin) was i.p. administered. Additionally, for surgery and intravitreal (ivt) injections, a drop of local analgesic, oxybuprocaine hydrochloride (0.4%, Unicaïne, Théa, Wetteren, Belgium) was applied to the treated eye. During recovery from anaesthesia, an ocular ointment (Tobrex; Alcon, Puurs, Belgium) was applied on the cornea to prevent corneal desiccation. All animals were sacrificed with an i.p. injection of an overdose of sodium pentobarbital (30 mg/kg, Nembutal).
All animal experiments were approved by the Institutional Ethical Committee of KU Leuven (P260/2014) and followed the guidelines of the Association for Research in Vision and Ophthalmology and the European Communities Council Directive of 22 September 2010 (2010/63/EU).
All animal experiments were approved by the Institutional Ethical Committee of KU Leuven (P260/2014) and followed the guidelines of the Association for Research in Vision and Ophthalmology and the European Communities Council Directive of 22 September 2010 (2010/63/EU).
3
Anesthesia and Euthanasia Protocol
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Surgical procedures were performed under general anesthesia (75 mg/kg ketamine, Anesketin, Eurovet; 1 mg/kg medetomidine, Domitor, Pfizer), which was reversed by atipamezole (1mg/kg atipamezole, Antisedan, Pfizer). In addition, a drop of local anesthesia (oxybuprocaïne 0.4%, Unicaïne, Théa) was given before the surgery and an antiobiotic ointment (tobramycin 0.3%, Tobrex, Alcon) was applied afterwards. All animals were sacrificed with an overdose of sodium pentobarbital (60 mg/kg, Dolethal, Vetoquinol), followed by cervical dislocation.
4
Retinal Layer Thickness Analysis Using SD-OCT
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Thickness of the retinal layers was evaluated using a spectral domain optical coherence tomography (SD-OCT) system (Envisu R2210, Bioptigen) (Buys et al., 2013) . Upon general anesthesia (i.p. 75 mg/kg body weight ketamine, Anesketin, Eurovet; i.p. 1 mg/kg medetomidine, Domitor, Pfizer), pupils were dilated by topical application of 0.5% tropicamide (0.5% Tropicol, Thea). SD-OCT was performed using 100 consecutive B-scan lines composed of 1000 A-scans, in a 1.4x1.4mm field. After the procedure, anesthesia was reversed by means of atipamezol (i.p. 1 mg/kg, Antisedan, Pfizer) and antibiotic ointment was applied to the eye (tobramycin 3 mg/g, Tobrex, Alcon). Total retinal thickness and thickness of different retinal layers were analyzed using InVivoVue Diver 2.2 software (Bioptigen).
5
Surgical Procedures under Anesthesia
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All surgical procedures were performed under general anesthesia (i.p. 75 mg/kg body weight ketamine, Anesketin, Eurovet; i.p. 1 mg/kg medetomidine, Domitor, Pfizer). After the procedure, anesthesia was reversed by means of atipamezole (i.p. 1 mg/kg, Antisedan, Pfizer), and antibiotic ointment (tobramycin 3 mg/g, Tobrex, Alcon) was applied to avoid corneal desiccation and infection of the eye. In order to exclude contralateral effects, which have been reported, for example, for GFAP expression and microglia reactivity [34 (link)–36 (link)], untreated eyes from a separate cohort of untreated animals served as controls.
6
Assessing Retinal Changes in Mice
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All experiments followed the ARVO and European Union guidelines for the use of animals in research and were approved by the Ethical and Animal Studies Committee (University of Murcia [UM]). Adult female albino Swiss mice (25–30 g) were obtained from Charles River Laboratories (L'Arbresle, France) and housed at the UM animal facilities in temperature and light controlled rooms (12-hour light/dark cycle) with food and water ad libitum. For surgical or animal manipulations, mice were anaesthetized with an intraperitoneal (ip) injection of ketamine (70 mg/kg Ketolar; Pfizer, Alcobendas, Madrid, Spain) and xylazine (10 mg/kg Rompun; Bayer, Kiel, Germany). Topical ointment (Tobrex; Alcon-Cusí, S.A., El Masnou, Barcelona, Spain) was applied during recovery to prevent corneal desiccation. Mice were euthanized with pentobarbital (Dolethal, Vetoquinol; Especialidades Veterinarias, S.A., Alcobendas, Madrid, Spain). In the present experiments we have used six naïve untouched mice, and 110 subjected to LIP; their left eyes were used as experimental while the right eyes served as controls. Because our previous studies characterizing the cone population of adult mice were done in female mice,55 (link) for comparison we have used only female mice in the present study.
7
Anesthesia and Euthanasia Protocol for Swiss Mice
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The experiments were performed on adult male albino Swiss mice (between 40 and 45 g) obtained from the breeding colony of the University of Murcia (Murcia, Spain). The animals were housed in temperature- and light-controlled rooms with a 12 hours light/dark cycle and ad libitum access to food and water. Light intensity within the cages ranged from 9 to 24 lux. All surgical procedures were performed under general anesthesia induced with an intraperitoneal (ip) injection of a mixture of ketamine (75 mg/kg, Ketolar®, Parke-Davies, Barcelona, Spain) and xylazine (10 mg/kg, Rompún®, Bayer, Barcelona, Spain). During recovery from anesthesia, the mice were placed in their cages and an ointment containing tobramycin (Tobrex®; Alcon, Barcelona, Spain) was applied to the cornea to prevent corneal desiccation and infection. Additional measures were taken to minimize discomfort and pain after surgery. The animals were killed with an ip overdose of pentobarbital (Dolethal Vetoquinol®, Especialidades Veterinarias, Alcobendas, Madrid, Spain).
8
Induction and Management of Ocular Hypertension
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The IOP was measured after sedating the animals with inhalation anesthesia using 2% isoflurane in oxygen (ISOFLO Isoflurane 100% p/p, Zoetis SL, Alcobendas, Madrid, Spain). OHT was induced under general anesthesia using an intraperitoneal injection of a mixture of ketamine (75 mg/kg; Anesketin®, Dechra Veterinary Products SLU, Barcelona, Spain) and medetomidine (0.26 mg/kg; Medetor®, Virbac España S.A., Barcelona, Spain). To prevent corneal desiccation and infection after the laser procedure, tobramycin (Tobrex®; Alcon, Barcelona, Spain) ointment was used. After OHT induction, all the appropriate procedures were adopted to mitigate the pain and discomfort of the animals. The animals were finally sacrificed after the intraperitoneal injection of an overdose of pentobarbital (Dolethal Vetoquinol®, Especialidades Veterinarias, Alcobendas, Madrid, Spain).
9
In Vivo Mouse Eye Surgical Procedures
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Adult pigmented C57Bl/6 male mice (25 g) were obtained from the University of Murcia breeding colony. All animals were treated in compliance with the European Union guidelines for Animal Care and Use for Scientific Purpose (Directive 2010/63/EU) and the guidelines from the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research. All procedures were approved by the Ethical and Animal Studies Committee of the University of Murcia, Spain (number: A1320140704).
Animals undergoing surgery were anesthetized by intraperitoneal injection of a mixture of ketamine (60 mg/kg; ketolar, Pfizer, Alcobendas, Madrid, Spain) and xylazine (10 mg/kg; Rompum, Bayer, Kiel, Germany). Analgesia was provided by subcutaneous administration of buprenorphine (0.1 mg/kg; Buprex, Buprenorphine 0.3 mg/mL; Schering-Plow, Madrid, Spain). During and after surgery, the eyes were covered with an ointment (Tobrex; Alcon S.A., Barcelona, Spain) to prevent corneal desiccation. Animals were sacrificed with an intraperitoneal injection of an overdose of sodium pentobarbital (Dolethal, Vetoquinol; Especialidades Veterinarias, S.A., Alcobendas, Madrid, Spain).
Animals undergoing surgery were anesthetized by intraperitoneal injection of a mixture of ketamine (60 mg/kg; ketolar, Pfizer, Alcobendas, Madrid, Spain) and xylazine (10 mg/kg; Rompum, Bayer, Kiel, Germany). Analgesia was provided by subcutaneous administration of buprenorphine (0.1 mg/kg; Buprex, Buprenorphine 0.3 mg/mL; Schering-Plow, Madrid, Spain). During and after surgery, the eyes were covered with an ointment (Tobrex; Alcon S.A., Barcelona, Spain) to prevent corneal desiccation. Animals were sacrificed with an intraperitoneal injection of an overdose of sodium pentobarbital (Dolethal, Vetoquinol; Especialidades Veterinarias, S.A., Alcobendas, Madrid, Spain).
10
Tobramycin 0.3% Ophthalmic Solution
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tobramycin 0.3% (Tobrex®, sterile ophthalmic solution, 5 ml, each ml contains tobramycin 3 mg, Alcon-Couvreur NV, Puurs, 2870, Belgium).
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