Nitric oxide levels in cochlear fluids of guinea pig 1

PURPOSE: To describe a technique to obtain guinea pigs cochlear fluids and measure nitric oxide (NO) concentration. METHOdS: Six guinea pigs were used and sacrificed. The cochlear fluids collected for measurement of NO, performed by chemiluminescence (NOA 280). RESULTS: Through the chemiluminescence was possible to analyze the concentration of NO in cochlear fluids obtained. Average levels of nitric oxide from guinea pigs was 12.55 μM. CONCLUSION: It is possible to obtain nitric oxide cochlear fluids, with this technique and nitric oxide concentration measure by chemiluminescence, a quantitative and more precise method.


Introduction
The search for an experimental model that provides a detailed and reliable study of the physiopathology of a wide range of human diseases is a constant challenge for scientists.The ideal model must reproduce these mechanisms, being at the same time, reproducible and easily executed.Low cost is another important characteristic for its feasibility worldwide.
In Otology, especially in the study of sensorineural hearing loss, experimental models are of utmost importance.Such models have been widely used and they resort to many technical resources and different species of animals.Among all experimental animals, guinea pig is preferred due to its biological resistance in captivity.Apart from that, its anatomy favors an adequate accessibility to the middle and inner ear via bulla mastoidea, which is well developed in guinea pigs 1,2 The main cause of sensory hearing loss is the injury in ciliated cells, and, the role of free radicals in producing cell lesion is gaining prominence for the evidences that lead to its participation in several pathological situations such as ototoxicity, hearing loss and cochlear ischemia [3][4][5] .
Most studies target the analysis of Nitric Oxide (NO), a free radical, produced by the action of nitric oxide synthase (NOS), its stable metabolites are nitrite and nitrate 6 .There are two main forms related to NOS: constitutive, whose activation depends on the calmodulin calcium complex, with two isomers called NOS I or neuronal NOS (nNOS) and NOSIII or endothelia NOS (eNOS) and a second one called inducible NOS (iNOS) or NOSII, whose activation is related to endotoxins and cytokines [6][7][8] .
Cochlear nitric oxide was demonstrated by Fessenden et al. 2% lidocaine solution with vascular constrictor (Figure 1).
Following the anesthesia an incision in the retro auricular region was performed with n° 15 scalpel (Figure 2) and the dissection in planes with iris scissor allowed the exposition of bulla mastoidea (Figure 3).Bulla mastoidea was opened with a high speed motor and a 2mm drill, under optical microscope (Figure 4), to meet the round window niche (Figure 5).The cochlear fluid was aspirated with a micropipette and an adapted insulin needle (Figure 6) by perforating the round window membrane (Figure 7).The fluid was kept in appropriate recipients under 0°C temperature.The same surgical procedure was performed on the other ear and the animals were sacrificed at the end of the experiment.
Despite the low negative pressure produced by the micropipette, its association with the manipulation of an insulin needle in the tympanic ramp presents a potential risk for membranous labyrinth membranes rupture.Therefore, the final aspirated product from the round window is probably a mixture of endolymph and perilymph, and hence named herein "cochlear fluids".A minimum of 10µL is needed for this measurement which is exactly the maximum volume that is obtained from the internal ear of these guinea pigs.Therefore, fluid from both ears was used to have this dosage performed.

Results
The final volume of cochlear fluid collected from each ear varied between 5 and 6 µM.At least 10 µM of fluid was needed to perform a reliable NOA analysis, therefore fluids from both ears were collected to have a satisfactory NO count.As the estimated volume of cochlear fluids (endolymph and perilymph) in guinea pig is 8.5 µM 12 in this experimental work the volume of cochlear fluid collected was about 60 to 70% of the total volume of the cochlear fluids.
NOA showed an average of 12.55 µM of nitric oxide in cochlear fluids of guinea pigs (Table 1).

Discussion
Experimental models that can simulate tissue and cellular complex physiological mechanisms, enabling the evaluation of the molecular activities of these structures, constitute currently the paradigms of the experimental models.
They are very important to scientific research, because through them it is possible to control multiple variables and a wide range of studies can be done.Studies about efficiency and toxicity of medicines, physiopathology of certain conditions, follow-up of surgical intervention, are the main applications of these models 13 .
In Otology, many researchers developed experimental studies using guinea pigs (Cavia porcellus).This animal is preferably used due to the fact that its anatomy favors inner and medium ear surgical procedures.Wysocki 2 in a recent work has described the guinea pig's anatomy in details facilitating the study of an access to the structures of the cochlear vestibular system.
Many inner ear diseases such as ischemia, acoustic trauma and ototoxic medicine damage may cause irreversible 3,5,8 .
NO role related to cellular injury process is being the target of countless studies.Production of NO in the inner ear is well documented, and it is well-known that the physiological amount of this free radical mean no harm.Lesion occurs when there is an NO overproduction that is not followed by an increase in anti free radicals 10 .
NO measurement in experimental studies constitutes a challenge because of its high volatility.Most of the studies measure its stable metabolites such as the nitrite and nitrate, or their conversion enzymes.An example of this statement is the work done by Ruan et al. 15 studying NO action in normal and ischemic cochlea of guinea pigs.They have used donors of NO such as S-nitrocysteine and nitroglycerin, as well as NO inhibitors such as N-nitro-L-arginine methyl ester.These authors infused either the NO donor or its inhibitor in the perilymphatic space through two little openings made in the basal gyrus of the cochlea, the remainder perilymph was drained by an additional opening.
Guinea pigs were sacrificed two days later and cochleae were taken for study.Some of them were artificially induced into the state of hypoxia and others were submitted to NO count.Hypoxia was performed in a closed environment without oxygen by exposing the cochlea for 20 to 120 minutes.Measurement was done using Griess colorimetric assay.Perylymph nitrite and nitrate were mixed to Griess reagent and their absorbance was registered in a 540nm wave spectrophotometer.Absorbance was compared to a pre established standard curve that showed NO values for the sample 15 .It is an indirect method to measure NO and the authors showed that in ischemic cochlea there is a NO decrease and the administration of exogenous NO could protect guinea pigs inner ear.
NO can also be measured by using its conversion enzyme as cited by Heinrich et al. 16 .They described a NO increase after a transtympanic injection of gentamicin in guinea pig cochlea.
After five days of drug administration animals were sacrificed and cochleae were prepared for histological and immunochemistry evaluation using anti-eNOS antibody.Gentamicin treated animals had a high level of cochlear injury together with an increase in conversion enzyme eNOS 16 .The advantage of this method is the possibility of distinguishing which conversion enzyme was increased; it is known that in normal conditions there is no inducible form of NOS in the cochlea, an anti iNOS antibody can be used to identify any increase in NO levels in the inner ear.
Takumida et al. 17  which is brighter, as it is higher is the intracellular NO.
In our study the chemoluminescent method was used to measure NO levels, since it is more sensitive to the slightest variation of NO quantity.This technique was used by Chen et al. 19 to demonstrate that the effects caused by hearing trauma can produce NO in the lateral wall of the cochlea.Guinea pigs were submitted to 40 hours of 105 dBSPL acoustic trauma, and their hearing was then evaluated by means of short latency evoked potential after zero, seven, 28 and 56 days.Following the last evaluation the animals were sacrificed, the cochlea removed and the otic capsule exposed.Lateral wall, stria vascularis and spiral ligament were removed by micro dissection, homogenized, and centrifuged.The supernatant was then analyzed by NOA.NO levels were increased after exposition to noise, and lingered for 28 days, decreasing to normal levels after 56 days.Hearing evaluation showed an increase of the hearing thresholds during the first days, decreasing after some days, but normal levels were not reached 18,19 .
There was no reference in the literature about the direct measurement of NO in aspirated cochlear liquids.The term "cochlear fluids" are here preferentially used because even though the approach being done by the round window.We believe that the negative pressure given by the micropipette and the manipulation of the needle would lead to a rupture of the tender membranes of the membranaceous labyrinth and the final aspirated product is probably a mixture of endo and perilymph.
The technique used in this study to obtain cochlear fluids was efficient, and enabled the NO concentration measurement.
Our technique stands out due to its feasibility and its application to many other studies such as ototoxicity, hearing trauma, and others.
Furthermore, its cost is low enough to facilitate research in this area, and it does not need special dyes or fixatives.
The only one difficulty found was the small amount of cochlear fluids obtained by aspiration, collection from both cochleae were needed so as to have NO measured.We believe that can be improve together with the improvement of aspiration technique.

Conclusions
This study demonstrates the feasibility of collecting cochlear fluids in vivo and the possibility to dose nitric oxide level.
This cost effective experimental model may contribute to future studies dealing with nitric oxide and inner ear.

FIGURE 1 -
FIGURE 1 -Guinea pig in right lateral decubitus to approach the left ear.

FIGURE 2 -
FIGURE 2 -Retro auricular incision of the skin.

FIGURE 5 -
FIGURE 5 -Identification of round window niche.

FIGURE 6 -
FIGURE 6 -Detail of the insulin needle.

FIGURE 7 -
FIGURE 7 -Aspiration of cochlear fluid through the round window.
published the first work of 'NO direct measurement', using a substance called 4.5-diaminofluorescein diacetate (DAF-2DA).This substance is able to cross the plasmatic cells membrane and once in the cytoplasm, it is hydrolized into a new compound called 4.5-diaminofluorescein (DAF-2).DAF-2 reacts with the existing intracellular NO, giving rise to triazolfluorescein (DAF-2T), that is a fluorescent green indicator,

TAbLE 1 -
Nitric oxide levels in guinea pigs cochlear fluids.