Method for lifelong detecting acute phase of staphylococcal otitis media in experiment

FIELD: experimental medicine.

SUBSTANCE: on the 3d - 4th d of experimental staphylococcal acute otitis media it is necessary to examine lactoferrin concentration in nasal mucosa and activity of beta-lysines in blood serum in experimental rabbits and at lactoferrin concentration being 119.8 ± 1.6 ng/ml (standard being 86.2 ± 1.4 ng/ml) and activity of beta-lysines being 69.4 ± 0.4% (standard being 54.8 ± 1.7%) it is possible to conclude on acute phase of inflammatory process in patient's middle ear.

EFFECT: higher accuracy of detection.

1 tbl

 

The invention relates to the experimental section of medicine, to methods of in vivo determination of objective indicators of the severity of the inflammatory process in the middle ear in rabbits with experimental staphylococcal otitis media in research.

In patients with acute otitis media acute inflammatory process in the middle ear can be set according to the anamnesis, complaints, clinical symptoms and condition otoscopes picture [4]. Clinical manifestations of acute experimental staphylococcal otitis media (ESOO) in laboratory animals is not clearly defined and do not allow to reliably determine the acute phase of the inflammatory process in the middle ear.

VA Debt has worked with rabbits, which was caused by acute experimental staphylococcal otitis media, and noted that the acute phase of the disease can be installed by conducting pathological studies of the mucous membrane of the tympanic cavity (SOBP) [3].

The study of pathological changes in ABP in the dynamics of otitis (1, 2, 3, 4, 5, 6 and day 7) showed that the disease onset (1-2 day) inflammatory changes occurred only in the connective tissue and is expressed in the extension of the lumen of blood vessels, stromal edema and the infiltration of its cells. Most acute is especially process was reached on the 3rd and 4th day of ASOSA and manifested significant destructive changes not only in the connective tissue, but in the epithelium SOBP. On day 5-6-7 otitis media, along with the ongoing alterative changes in the connective tissue was observed proliferative processes in the epithelium of the mucous membrane.

However, to conduct such studies it is necessary to remove the animal from experience that is not always the same as the objective of the conducted experiment.

The novelty of the present invention is the determination of the acute phase of the inflammatory process in the middle ear in experimental animals by the level of lactoferrin in the nasal mucus and activity of beta-lysine in serum.

A significant difference of the invention is that the acute phase of the inflammatory process in the middle ear of rabbits with acute experimental staphylococcal otitis media, which falls on the 3rd or 4th day of the disease, determine in vivo, the level of lactoferrin in the nasal mucus and the degree of activity of beta-lysine in serum: when the concentration of lactoferrin, 119,8±1.6 ng/ml (normal - 86,2±1,4 ng/ml, P<0.01) and activity of beta-lysine 69,4±0,4% (the norm for healthy - 54,8±1,7%, P<0,01) judge the acute phase of the inflammatory process in the middle ear.

To compare the dynamics of pathomorphological changes in ABP with the level of immunological parameters (lactoferrin in the nasal mucus and beta-lysine in serum) conducted comprehensive research is Finance in models of acute experimental staphylococcal otitis media in rabbits.

The method consisted of 4 stages of the study:

1. Creating a model of acute staphylococcal otitis media in rabbits.

2. The study of pathological changes in ABP in the dynamics of ASOSA.

3. The concentration of lactoferrin in the nasal mucus to play otitis media and at different periods of its development.

4. Determination of the activity of beta-lysine in serum of healthy animals and in the dynamics of experimental otitis media.

Acute otitis media was called in 37 adult rabbits of the chinchilla breed, weighing from 2 to 3 kilos During playback of acute otitis media in animals for was based on the methodology Alhoceima (1966), but instead of dogs, we used to experience in rabbits [2]. In addition, we've simplified the process of playing otitis animals. Before the introduction of the infectious agent in the tympanic cavity, we did not make the amputation of the ear and was limited to one-time introduction 1.0 ml 5 billion mist daily culture of Staphylococcus aureus, which was quite sufficient for the development of acute inflammation in the middle ear. As the pathogen used aureus, given the large share of this organism in the etiology of acute middle ear infections [5]. And used laboratory strain of Staphylococcus aureus, isolated from a patient with acute suppurative otitis media the Control group consisted of 9 healthy animals.

Pathological changes in the mucous membrane of the tympanic cavity were studied in 27 rabbits with ESOSA and 9 healthy animals. In the first 7 days of ASOSA these studies were conducted daily, then at 18 and 30 day. To obtain SOBP experimental animals were derived from experience by introducing 5 cm3air into the ear vein, after which operations were performed on the middle ear with the separation and removal SOBP. The obtained tissue was fixed in 10% neutral formalin and after 24 hours were filled with wax. Historty thickness of 6-8 μm, manufactured on a rotary microtome, after deparaffinize stained with Mayer hematoxylin and eosin. Research was conducted on the optical level with increasing ×280, ×630.

Immunological indicators: the level of lactoferrin in the nasal mucus and the activity of beta-lysine in serum were studied in 9 healthy animals before pathomorphological research SOBP and 37 - experimetally otitis media in the dynamics (1 day 3-4 day 6-7 day, 18 and 30 days). And 27 rabbits immunological parameters were studied in a specified time before pathomorphological studies SOBP and 10 - vivo during the development and course of ASOSA.

The determination of the content in the nasal mucus levels of lactoferrin, which is an important marker vospalitelno the process, chosen by chance. Middle ear and nasal cavity - two connected (through the auditory tube) border of the anatomical Department. There is information indicating the status of the upper respiratory tract and, in particular, the nasal cavity and nasopharynx in the etiology of middle ear infections [7, 9]. It is natural to assume the reverse: the impact of the dynamics of the inflammatory process in the middle ear on the nature of changes in indicators of nonspecific protection (lactoferrin) in the slimy contents of the nasal cavity.

Quite informative test that reflects the nature of the inflammatory process in the body, is an indicator of the activity of beta-lysine in serum [8].

The concentration of lactoferrin in the nasal mucus was determined by enzyme immunoassay using test systems "Vector-best" (Russia) and has been referred to in ng/ml [6]. Mucus from the nose was obtained by leaching it from the left and right nostrils rabbits 4 ml of sterile saline solution. The activity of beta-lysine in serum was determined fotokatalitichyeskim method (Avebury, Bahramov, Apoda, 1972) and was expressed in percent [1]. Blood for the study was received from the heart in the amount of 3-4 ml prior to infection and at different periods of development of experimental otitis media.

In healthy rabbits, the mucous membrane of the tympanic cavity tone is Aya and presents connective tissue. The concentration of lactoferrin in the nasal mucus was 86,2±1,4 ng/ml, the activity of beta-lysine in serum -54,8±1.7% (table 1).

All infected rabbits on achedule day developed the clinic of acute otitis media, which appeared troubled by the behavior of the animal, scratching paw patient ear, hyperemia and infiltration of the mucous membrane of the tympanic cavity and the remaining portion of the membrane. The structure of the epithelium was not changed, but in the connective tissue appeared dilated blood vessels. The level of lactoferrin in the nasal mucus was equal to 87.8±1.8 ng/ml, the activity of beta-lysine in serum was 60,3±1,9%.

The greatest development of the inflammatory process in the middle ear reached for 3-4 days. The mucous membrane of the tympanic cavity was dramatically swollen with pronounced symptoms of destruction of epithelium and connective tissue. Infiltration of connective tissue, a large number of cells caused delamination of the fibers and their death. The concentration of lactoferrin in the nasal mucus and the activity of beta-lysine in serum were respectively: 119,8±1.6 ng/ml (P<0.01) and 69,4±0,4% (P<0,01).

On the 6-7th day of the disease the severity of the inflammatory process in the middle ear in experimental animals decreased. In the midst of inflammation epithelium was killed on the periphery proliferated, because h is th acquired the multilayered structure. The concentration of lactoferrin was equal 92,2±3.6 ng/ml, the activity of beta-lysine - 59,0±1,8%.

On 18 and 30 day otitis inflammation in the middle ear has acquired the character of a subacute course: the epithelium was clad in connective tissue with fibroblasts was noted a large number of lymphoid elements and plasma cells. Indicators of activity of lactoferrin in the nasal mucus on these terms were respectively equal to 93.5±2.8 ng/ml and 94,4±1.9 ng/ml, beta-lysine: 58,4±3,4% and 59.3±2,9%.

The results of the study showed that the acute phase of the inflammatory process in the middle ear, manifested by the marked destruction of the epithelium and connective tissue of the mucous membrane of the tympanic cavity on the 3rd or 4th day of the disease, significantly characterized by a high concentration of lactoferrin in the nasal mucus and allows you to use these immunological parameters in determining the acute phase of otitis media in the experiment.

The positive effect of this method is that the acute phase of the inflammatory process in the middle ear in experimental staphylococcal otitis installed in vivo. by the highest level of lactoferrin in the nasal mucus and activity of beta-lysine in serum.

Literature

1. Bukharin O.V., Frolo the B.A., Lud A.P. Rapid method for the determination of beta-lysine in serum //Journal of Microbiology, epidemiology and immunology. 1972, No. 9. - P.42-44.

2. Gottlieb AL Eksperimentalnaya model of chronic suppurative inflammation of the middle ear in dogs //Sat. proceedings of the Leningrad scientific research Institute of ear, throat, nose and speech. L., 1966, t - S-157.

3. Debts, VA Pathological changes in the mucous membrane of the tympanic cavity in experimental otitis media //Collection of scientific works. Orenburg. 2000 - P.53-55.

4. Mechaniken NV Acute inflammation of the middle ear //Manual of otorhinolaryngology. M., Moscow. 1994 - Pp.92-101.

5. Mironov, A., Savitskaya SURDS, A. Vorobyev, Nesterov M.B. Microflora in diseases of ENT organs and nervous system in patients of the Moscow region //Bulletin of otorhinolaryngology. 2001. No. 4 - P.31-35.

6. Officers V.I., Yurkin E.A., Tkachenko so-CALLED. Lactoferrin - strip //instructions for use. Novosibirsk - 2002, 23C.

7. Rosenfeld IM an Acute inflammation of the middle ear in adults and children. Mastoiditis. Antrim. Petroset. // Manual of otorhinolaryngology. M, Moscow, 1960 - Vol.2 - P.137-211.

8. Sugareva TI Characteristic factors of natural resistance in staphylococcal pneumonia. Abstract. dis.... Kida. the honey. Sciences. - Chelyabinsk - 1977 - 13 C.

9. Tarasov DI, Fedorova H.K., V.P. Bykov Diseases of the middle ear. - M, Moscow. 1988.- 286 C.

The method for determining the acute phase staph otitis media in the experiment, including studies of animals in the dynamics, characterized in that 3-4 day otitis lifetime investigate the concentration of lactoferrin nasal mucus and the activity of beta-lysine in serum and the concentration of lactoferrin 119,8±1.6 ng/ml, normal 86,2±1,4 ng/ml, and the activity of beta-lysine 69,4±0,4%, the rate of 54.8±1.7 per cent, judge the acute phase of the inflammatory process in the middle ear.



 

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