Action of Ropivacaine as a Surface Anaesthetic on the Cornea of Rabbits
N. Jagan Rao
The aim of the present study was to investigate the role of Ropivacaine as a surface anesthetic on the cornea of rabbits. Twenty Albino rabbits (weighing 2.5 to 3.0 kg of either sex) were randomly allocated to 2 treatment groups-Group I and II. The upper and lower eye lashes of all the rabbits were carefully clipped off. The conjunctival sac of right eye was held open to form a pocket. Into these pockets, animals in Group I were delivered 1 drop (containing 535.71 μg of Ropivacaine) of the standard solution of Ropivacaine and those in Group II were delivered 2 drops (containing 1071.42 μg of Ropivacaine) of the same solution. The left eye served as the control. The corneal reflex was elicited by touching the cornea from the side using a cotton wisp. The time between the disappearance and reappearance of corneal reflex (duration of action) was registered. Statistical analysis revealed highly significant differences between group I and group II (p<0.001). The mean onset of action for group I was 10 min and for group II was 5 min. The mean duration of action for group I was 29±4.595 and for group II was 48±5.869. Ropivacaine can be used as surface anaesthetic as Proparcaine and Tetracaine for removal of foreign bodies and other clinical conditions in ophthalmology.
Received: September 21, 2010;
Accepted: February 22, 2011;
Published: March 16, 2011
Surface anesthesia or anesthesia of mucous membranes of the nose, mouth, throat,
tracheobronchial tree, esophagus and genitourinary tract can be produced by
direct application of aqueous solutions of salts of many local anesthetics or
by suspension of the poorly soluble local anesthetics. Tetracaine (2%), lidocaine
(2 to 10%) and cocaine (1 to 4%) are typically used. The local anesthetics are
absorbed rapidly into the circulation following topical application to mucous
membranes or denuded skin (Catterall and Mackie, 2006).
Cardiac toxicity of bupivacaine stimulated interest in developing a less toxic
and long-lasting local anesthetic. One result of the search was the development
of the amino ethylamide Ropivacaine (Catterall and Mackie,
2006). It is a newer bupivacaine congener which is superior to bupivacaine
for epidural anesthesia because of its decreased potency for motor block. It
has been recently approved for use in adults. It is less toxic to the central
nervous system and heart and interferes less with motor function than bupivacaine
(Lonnqvist et al., 2000). The S-enantiomer was
chosen because it has a lower toxicity than the R-isomer (Catterall
and Mackie, 2006). It blocks Aβ and C fibres (involved in pain transmission)
more completely than Aβ fibres which control motor function. Though equi-effective
concentrations of Ropivacaine are higher than those of Bupivacaine, a greater
degree of separation between sensory and motor block has been obtained with
epidural Ropivacaine (Tripathi, 2008).
Ropivacaine is the S-enantiomer of 1-propyl-2, 6-pipecoloxylidide
(Catterall and Mackie, 2006). It is a new, long-acting
local anaesthetic of the amide type. It contains a single chiral centre and
is used as the pure S-(-) enantiomer. In vivo racemization does not occur
after systemic administration of the drug. Its physiochemical properties includes-wt.
274.4 (base), pK 8.1 and log D (pH 7.4 n-octanol vs. buffer) 2.15. Intravenous
Ropivacaine shows linear pharmacokinetics and the drug is almost completely
metabolized, with less than 1% of the dose excreted unchanged (Arlander
et al., 2003).
Several studies have been done to reveal the scope of Ropivacaine for use in
various diseases and conditions. Infiltration of split skin grafts donor site
with Ropivacaine improves postoperative pain during 48 h. This is a safe and
efficient method to improve comfort in addition to a standardized occlusive
dressing (Trost et al., 2005). Dural surface
area influences the spread of epidural anesthesia with Ropivacaine and posterior
fat volume influences the duration of epidural anesthesia in healthy patients
within a narrow age range. Epidural venous plexus velocity might also influence
the duration of epidural anesthesia with Ropivacaine (Higuchi
et al., 2004). Topical anesthesia with Ropivacaine is safe and effective
in pterygium surgery (Caccavale et al., 2010).
Ropivacaine proved to be effective for pain relief after hernia repair in ilioinguinal
blocks accompanying general anesthesia (Wulf et al.,
Ropivacaine has efficacy similar to lidocaine, with slightly longer onset and
duration of the motor blockade. In addition, Ropivacaine (0.75%) induces lower
intraocular pressure and less pain on injection than does lidocaine (2%) when
used in peribulbar anesthesia for cataract surgery (Olmez
et al., 2004). Compared with lidocaine, intravenous regional anesthesia
with Ropivacaine appears to be comparable but has longer-lasting residual anesthesia
(Chan et al., 1999). Topical Ropivacaine performed
at least as well as topical lidocaine in efficacy and safety in cataract surgery.
It provided sufficient and long-lasting analgesia without the need for supplemental
intracameral anesthesia in most cases (Martini et al.,
The 0.5% Ropivacaine with 1:200,000 epinephrines is equivalent to 0.5% bupivacaine
with 1:200,000 epinephrines in pharmacologic action. The duration of pulpal
anesthesia is less for Ropivacaine without epinephrine. Ropivacaine with epinephrine
has the potential to replace bupivacaine with epinephrine in clinical dental
practice because of the decreased potential for cardiac and central nervous
system toxicity (Kennedy et al., 2001).
However, the action of Ropivacaine as a surface anesthetic has not been studied so far. The aim of the present study was to investigate the role of Ropivacaine as a surface anesthetic on the cornea of rabbits.
MATERIALS AND METHODS
The study was performed between 5/2/2010 to 24/6/2010 for a period of four months at Mamata Medical College (MMC), Khammam Andhra Pradesh (AP). Twenty albino rabbits (weighing 2.5 to 3.0 kg of either sex) were obtained from the Research Animal House of MMC and used in the study. The animals were housed at a temperature of 24±2°C and relative humidity of 30-70%. All animals had free access to water and standard pelleted laboratory animal diet. All the experimental procedures and protocols used in this study were reviewed and approved by the Institutional Animal Ethical Committee of MMC, Khammam, AP and were in accordance with the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA).
Calculation of doses from standard solution of ropivacaine: Commercially available standard solutions of Ropivacaine containing 0.75% Ropivacaine in 1 mL of solution (7.5 mg mL-1) were used to prepare the test solutions.
One milliter of solution contains 7.5 mg or 7500 μg of Ropivacaine.
One milliter of solution contains 14 drops.
Thus, 1 drop contains 7500/14 = 535.71 μg of Ropivacaine.
Two drops contains 535.71x2 = 1071.42 μg of Ropivacaine.
Delivering the test solutions to experimental groups: The animals were placed in rabbit holding cages and were randomly allocated to 2 treatment groups-Group I and Group II. The upper and lower eyelashes of all the rabbits were carefully clipped off to avoid the corneal reflex initiated by accidental touching of the eyelashes. The conjunctival sac of right eye was held open to form a pocket. Into these pockets, animals in group I were delivered 1 drop (containing 535.71 μg of Ropivacaine) of the standard solution of Ropivacaine and those in Group II were delivered 2 drops (containing 1071.42 μg of Ropivacaine) of the same solution. The left eye served as the control.
The corneal reflex was elicited by touching the cornea from the side using a cotton wisp. The test was started 5 min after the application of the drug in both groups and repeated every 5 min until the corneal reflex was lost (blinking lost) followed by reappearance of the corneal reflex (blinking reappears). The time between the disappearance and reappearance of corneal reflex (duration of action) was registered.
The results obtained were recorded and tabulated. All data was expressed in terms of a Mean±Standard deviation. To compare the intergroup differences for onset of action of the anesthetic and the duration of action of the anesthetic, a student t test was done using the software Primer for Biostatistics Version 5.0. All probability values less than 0.05 (p<0.05) were considered significant and those less than 0.001 (p<0.001) were considered highly significant.
The time of disappearance of corneal reflex and the time between the disappearance
and reappearance of corneal reflex were recorded in 20 albino rabbits after
the administration of two doses of a standard solution Ropivacaine. At baseline
(0 min), all the rabbits in both group I and II (n = 20) showed the presence
of a normal corneal reflex. Five min after the application of the test solution,
all rabbits in-group I still retained a normal corneal reflex while that in-group
II lost their corneal reflex (Table 2). By 10 min, group I
rabbits also had lost their corneal reflex (Table 1).
||Corneal reflex of group I rabbits (n = 10), Dose: 1 drop (535.71
|+: Corneal reflex present; : Corneal reflex absent
||Corneal reflex of group II rabbits (n = 10), Dose: 2 drops
|+: Corneal reflex present; : Corneal reflex absent
In the group I rabbits, 1 rabbit regained corneal reflex after 30 min (duration
of action = 25 min), 2 rabbits regained their corneal reflex after 35 min (duration
of action = 30 min), 5 rabbits regained their corneal reflex after 40 min (duration
of action = 35 min) and 2 rabbits regained their corneal reflex after 45 min
(duration of action = 40 min) (Table 1). In the group II rabbits,
2 rabbits regained their corneal reflex after 45 min (duration of action = 45
min), 3 rabbits regained their corneal reflex after 50 min (duration of action
= 50 min), 2 rabbits regained their corneal reflex after 55 min (duration of
action = 55 min) and 3 rabbits regained their corneal reflex after 60 min (duration
of action = 60 min) (Table 2). For the control sites (left
eye), the corneal reflex was positive throughout the period of the experiment.
Statistical analysis revealed highly significant differences between group I and II (p<0.001). The mean onset of action for group I was 10 min and for group II was 5 min (Table 3). The mean duration of action for group I was 29±4.595 and for II was 48±5.869 (Table 4).
|| Mean onset of action of two different doses of ropivacaine
|n: Sample size, SD: Standard deviation, SEM: Standard error
mean, *Statistically significant
|| Mean duration of action of two different doses of ropivacaine
|n: Sample size, SD: Standard deviation, SEM: Standard error
mean, *Statistically significant
Topical anesthetic agents used clinically in ophthalmology include Cocaine,
Proparcaine and Tetracaine drops and Lidocaine gel. Proparcaine and Tetracaine
are used for removal of foreign bodies, for tonometry and for superficial corneal
surgeries with no side effects (Catterall and Mackie, 2006).
Ropivacaine is a local anaesthetic agent mainly used for both epidural and regional
anesthesia. Side effects with topical Ropivacaine have not been studied extensively
but one study with impression cytology presents a non-invasive or minimally
invasive biopsy of the ocular surface epithelium with no side effects (Soker
et al., 2007). Ropivacaine has also been used safely and effectively
in pterygium surgery. The Long-lasting anesthesia with this agent permitted
performing the surgical procedures with autograft conjunctival graft and fibrin
glue to attach the flap with low pain perceived by the patients, low surgical
invasivity and short duration of surgery (Caccavale et
Recently, topical anesthesia and intracameral anesthesia have become popular
in modern cataract surgery. During the last decade, the use of Ropivacaine as
a research tool has experienced an enormous growth and has greatly contributed
to the understanding of ocular surface pathology, including investigation of
the toxic effects of anterior camera applied chemicals, for instance, application
of Ropivacaine to the corneal endothelium. However, some authors advise caution
with the use of intracameral anesthetic agents because of possible toxic effects
in intraocular structure, especially the corneal endothelium. Many experimental
studies were performed to investigate endothelial toxicity. One percent lidocaine
hydrochloride (HCL) causes a transient endothelial cell edema to the in vitro
perfused endothelium of human and rabbit corneas (Martini
et al., 2002).
Ropivacaine 1% and lidocaine 2% are safe and effective agents inpatients undergoing
phacoemulsification using a topical anesthesia. However, Ropivacaine provided
better operative conditions than lidocaine for the surgeon and comfortable surgical
circumstances for the patient (Nicholson et al.,
1999). Topical anesthesia with ropivacaine was safe, feasible and more effective
than lidocaine in cataract surgery (Borazan et al.,
2008). One of the studies showed experimentally that, corneas exposed to
0.01% Ropivacaine concentration in vitro manifested no serious damage histologically.
The impression cytology method can be used in the investigation of the toxic
effect of various intracameral anesthetic agents (Soker
et al., 2007). The efficacy of 1% ropivacaine for topical anesthesia
in dentistry was comparable with 20% benzocaine gel and eutectic mixture of
local anesthetics 2.5% lidocaine and 2.5% prilocaine (Franz-Montan
et al., 2007). The intraperitoneal instillation of ropivacaine was
effective in reducing postoperative pain and in shortening the recovery course
after laparoscopic colectomy. The additional instillation of ropivacaine at
the end of the surgery proved even more effective (Baek et
al., 2010). In another study, they concluded that administration of
intraperitoneal ropivacaine reduced pain during the post-operative period after
laparoscopic appendectomy (Kang and Kim, 2010).
Ropivacaine is a pure S-enantiomer that is less lipid-soluble and less cardiotoxic
than bupivacaine but more cardiotoxic than lidocaine. Ropivacaine is the newest
long-acting, enantiomerically pure (S-enantiomer) amide local anaesthetic, designed
by modification of an existing one. Chemically, it is very similar to bupivacaine
and mepivacaine. All of these three anesthetics come from the family of molecules
known as pipecolyl xylidines which combine the piperidine ring of cocaine with
xylidine from lidocaine. Substitution of methyl, butyl and propyl groups on
the piperidine ring give rise to mepivacaine, bupivacaine and Ropivacaine, respectively.
The high level of potency and lipid solubility of Ropivacaine suggests a CNS
toxicity profile similar to that of bupivacaine. Studies on anaesthetized rats
showed that the cumulative doses of levobupivacaine and Ropivacaine that produced
seizures were similar and were larger than those of bupivacaine. The predicted
cardiac toxicity profile of Ropivacaine has been extensively studied and animal
studies confirm an arrhythmogenicity of Ropivacaine that is intermediate between
that of mepivacaine and bupivacaine. The cumulative doses of levobupivacaine
that produced dysrhythmias and asystole were smaller than the corresponding
doses of Ropivacaine, but they were larger than those of bupivacaine. Ropivacaine-induced
cardiac arrest was more susceptible to treatment than that induced by bupivacaine
or levobupivacaine (Lonnqvist et al., 2000; Soker
et al., 2007; Tripathi, 2008; Marron-Pena
and Rivera-Flores, 2008).
Another study on rats concluded that Ropivacaine, even at equipotent dose,
is less toxic than bupivacaine. In rabbits and pigs, an indication was found
that Ropivacaine is less cardiodepressive and arrhythmogenic than bupivacaine.
Ropivacaine, according to animal data, is less neurotoxic and cardiotoxic than
bupivacaine. Based on available clinical data, Ropivacaine appears to be as
active and well tolerated as bupivacaine, when equianalgesic doses are compared
and to block nerve fibres involved in pain transmission (A delta and C fibres)
to a greater degree than those controlling motor function (A beta fibres). The
greater degree of separation between motor and sensory blockade seen with Ropivacaine
relative to bupivacaine at lower concentrations (approximately 5 mg kg-1)
will be advantageous in certain applications (Nicholson
et al., 1999).
This study was done to see whether Ropivacaine produces surface anesthesia or not. This study shows that Ropivacaine is a potent surface anaesthetic of fast onset with intermediate duration of action with dose of one drop i.e., 187.5 μg the onset of action (loss of corneal reflex is 10 min) and duration of action (reappearance of corneal reflex is 29 min) as shown in Table 4. With 2 drops i.e., 375 μg of Ropivacaine dose the onset of action (loss of corneal reflex is 5 min) and duration of action (reappearance of corneal reflex is 48 min) as shown in Table 4.
Like Proparcaine and Tetracaine, Ropivacaine can also be used as surface anaesthetic for removal of foreign bodies and other clinical conditions in ophthalmology. Different studies show safety and potency of the drug as topical anaesthetic with minimal cardiac and neurotoxicities. However, further studies should be done with different doses of Ropivacaine and further comparative studies with lidocaine and bupivacaine should be done to elicit the potency of Ropivacaine as surface anaesthetic.
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