RU2766298C2 - Method of treating corneal burns - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to ophthalmology, and can be used for conservative treatment of corneal burns. Method involves instillation of an aqueous solution of lactoferrin 2.5 mg/ml into the conjunctival cavity according to the following scheme: in case of alkaline burn — 2 drops 5 times day for 10 days; for acid burn — 1 drop 3 times day for 14 days; for thermal burns — 1 drop 3 times day for 28 days.

EFFECT: use of the invention enables to increase the effectiveness of the treatment of corneal burns, accelerate the epithelization of corneal defects, reduce the degree of manifestation of the inflammatory reaction to the burn injury, accelerate the process of repairing the defects of the corneal epithelium and stroma, reduce the intensity and area of corneal opacity.

1 cl, 20 dwg, 3 tbl, 3 ex

Description

SUBSTANCE: invention relates to medicine, namely to pharmacotherapy of eye diseases, and can be used for conservative treatment of corneal burns.

Previous level

One of the most severe traumatic injuries of the organ of vision is a burn of the cornea. The prevalence of corneal burns depends on the degree of development of the state and production, and ranges from 11.5 to 22.1% of all eye injuries [Sharma, Namrata, Manpreet Kaur, Tushar Agarwal, Virender Singh Sangwan and Rasik B Vajpayee. Treatment of acute ocular chemical burns. Survey of ophthalmology. 2019; 63(2): 214-235]. More than 40% of victims of severe burns become visually disabled of 1-2 groups [Pargament JM, Armenia J, Nerad JA. Physical and chemical injuries to eyes and eyelids. // Clin Dermatol. 2015;33920:234-237. Chernysh V.F., Boyko E.V. Eye burns. State of the problem and new approaches. 2nd ed. add. Moscow: GEOTAR-Media; 2017. Kulikov A.N., Chernysh V.F., Churashov S.V. On the new classification of eye burns. //Bulletin of ophthalmology. 2020; 2:134-142. doi.org/10.17116/oftalma2020136021134].

According to the clinical guidelines for the treatment of eye burns published in 2020, all patients from the first days after a burn are recommended to use topical glucocorticosteroids, antibacterial and / or antiseptic agents, drugs that promote regeneration (Dexpanthenol), antioxidant agents and tissue repair stimulants (Methylethylpyridinol, Solcoseryl, deproteinized hemoderivated blood of calves). M-cholinolytics are recommended for patients with severe corneal syndrome and/or cyclitis. From the third day after a moderate to severe burn with a threat of ulceration, instillations and / or intravenous injections of fibrinolysis inhibitors are added to the treatment. In the case of an increase in intraocular pressure, antiglaucoma drugs are prescribed locally to patients to reduce intraocular pressure to the target level [http://avo-portal.ru/documents/fkr/odobr/burns.pdf].

An important factor leading to aggravation of tissue damage in burns is oxidative stress. Immediately after exposure to alkali on the corneal tissue, the synthesis of reactive oxygen species (ROS) increases, the antioxidant protection of the cornea cannot cope with their deactivation, and an excess of free radicals occurs. In turn, free radicals damage the membrane structures of cells, structural and functional proteins, as a result, corneal cells die, and burns become more severe [Cejkova J., Trosan P., Cejka C. et al. Suppression of alkali-induced oxidative injury in the cornea by mesenchymal stem cells growing on nanofiber scaffolds and transferred onto the damaged corneal surface// Experimental Eye Research. 2013. 116 (2): 312-323].

A known method for the treatment of chemical burns of the cornea (RU 2028133 C1). Before applications of lyophilized plasma on the cornea, silica gel is adsorbed from the surface of the cornea until it is moistened, after which it is washed off with saline, and manipulations are performed 1-2 times a day during the entire period preceding epithelialization.

Silica gel is fire and explosion-proof, according to the degree of impact on the body it belongs to substances of the 3rd hazard class, however, silica gel dust contains from 10 to 70% free silicon dioxide, chronic inhalation of such dust may develop lung disease - silicosis. Therefore, when working with silica gel on a regular basis, it is recommended to use individual respiratory protection against dust. This may be the reason for the limited use of silica gel in medical practice.

A known method for the treatment of eye burns (RU 2038088 C1). For treatment, a 0.5% solution of echinochrome is administered subconjunctivally.

However, the pain of manipulation is a limitation of the use of this method in a number of patients.

Since the use of antioxidant drugs for the treatment of eye burns is pathogenetically justified, the closest (prototype) is a method for treating eye burns (EN 2495651 C1). Instillations are carried out in the conjunctival cavity of eye drops of 2% Mexidol solution 2-4 times a day immediately after the burn and for the next two weeks.

However, mexidol does not have an antibacterial effect, and in the treatment regimen for burns, additional use of antibacterial drugs is necessary to prevent the onset of a secondary infection.

The technical solution is to reduce the inflammatory response, accelerate the epithelialization of the burn zone, prevent the development of corneal perforations and prevent the addition of a secondary infection.

The technical result is achieved by the fact that, as in the known method, an antioxidant compound is instilled into the conjunctival cavity.

A feature of the claimed invention is that an aqueous solution of lactoferrin 2.5 mg/ml is used as an antioxidant preparation according to the following scheme:

- with an alkaline burn - 2 drops 5 times a day for 10 days;

- with acid burn - 1 drop 3 times a day for 14 days;

- for thermal burns - 1 drop 3 times a day for 28 days.

The method is illustrated by a detailed description, examples of execution and illustrations, which show:

Fig. 1 - Rabbit cornea immediately after acid burn.

Fig. 2 - Rabbit cornea of group 1 1 day after acid burn.

Fig. 3 - The cornea of a rabbit of the 1st group 3 days after the application of an acid burn.

Fig. 4 - Rabbit cornea of the 1st group 5 days after the application of an acid burn.

Fig. 5 - Rabbit cornea of the 1st group 7 days after the application of an acid burn.

Fig. 6 - Rabbit cornea of the 1st group 14 days after the application of an acid burn.

Fig. 7 - Rabbit cornea of group 2 1 day after acid burn.

Fig. 8 - Rabbit cornea of the 2nd group 3 days after the application of the acid burn.

Fig. 9 - Rabbit cornea, group 2, 5 days after acid burn.

Fig. 10 - Rabbit cornea immediately after thermal burn.

Fig. 11 - Rabbit cornea of the 1st group 1 day after thermal burns.

Fig. 12 - Rabbit cornea of the 1st group 3 days after thermal burns.

Fig. 13 - Rabbit cornea of the 1st group 5 days after thermal burns.

Fig. 14 - Rabbit cornea of the 1st group 7 days after thermal burns.

Fig. 15 - Rabbit cornea of the 1st group 28 days after thermal burns.

Fig. 16 - Rabbit cornea of the 2nd group 1 day after thermal burns.

Fig. 17 - Rabbit cornea of the 2nd group 3 days after thermal burns.

Fig.18 - The cornea of a rabbit group 2 5 days after thermal burns.

Fig.19 - The cornea of a rabbit group 2 7 days after thermal burns.

Fig. 20 - Rabbit cornea of the 2nd group 28 days after thermal burns.

The method is carried out as follows.

Animals with a chemical burn of the cornea were treated with a solution of lactoferrin at a concentration of 2.5 mg / ml, 1-2 drops 3-5 times a day for the first 10-28 days, depending on the agent that caused the burn and its severity, and exactly:

- with an alkaline burn - 2 drops 5 times a day for 10 days;

- with acid burn - 1 drop 3 times a day for 14 days;

- for thermal burns - 1 drop 3 times a day for 28 days.

To study the effectiveness of the effect of local application of lactoferrin on the healing of a burn wound of the cornea, an experimental study was conducted.

EXAMPLE 1 Experimental simulation of moderate alkaline burn.

Research on female rabbits of the Chinchilla breed (weight - 2.0 - 2.2 kg). Experimental modeling of an alkaline burn of moderate severity was performed on 12 rabbits (24 eyes) weighing 2.0-2.2 kg. Alkaline burn was caused by application of filter paper in the form of a circle with a diameter of 10 mm, moistened with 2.5% sodium hydroxide solution with an exposure of 5 seconds to the cornea under local anesthesia with 0.4% inocaine. After removing the filter paper, the eyes were washed with 0.9% sodium chloride solution.

Experimental animals were divided into four groups - three experimental and one control:

- group 1: control group (3 rabbits; 6 eyes). Animals were instilled with isotonic (0.9%) sodium chloride solution (2 drops 5 times a day for 10 days);

- group No. 2: experimental group (3 rabbits; 6 eyes). The animals were instilled with lactoferrin at a concentration of 0.5 mg/ml (2 drops 5 times a day for 10 days);

- group 3: experimental group (3 rabbits; 6 eyes). The animals were instilled with lactoferrin at a concentration of 2.5 mg/ml (2 drops, 5 times a day for 10 days);

- group 4: experimental group (3 rabbits; 6 eyes). The animals were instilled with lactoferrin at a concentration of 5.0 mg/ml (2 drops 5 times a day for 10 days).

The clinical study included examination of the anterior segment of the eyes using focal and lateral illumination. Animals were observed daily for 10 days. The condition of the eyes was assessed according to the following criteria: the severity of the inflammatory reaction, the size of the corneal defect, the intensity of corneal opacity.

The degree of the inflammatory reaction was assessed visually according to the scoring system:

- 0 points - no inflammatory reaction;

- 1 point - a slight mixed injection of the conjunctiva, poor discharge, no edema of the corneal tissue surrounding the burn;

- 2 points - mixed injection of the conjunctiva, moderate mucopurulent discharge, swelling of the walleye and the surrounding corneal tissue;

- 3 points - pronounced mixed injection of the conjunctiva with chemosis, abundant mucopurulent discharge, severe corneal edema.

The size of the epithelial defect in mm ^{2 of} the cornea was estimated by the area of the epithelial defect after instillation of fluorescein (1% solution; from 3 to 5 drops) by measuring the horizontal and vertical meridians with a calibrometer.

The intensity of corneal opacity in the burn area was assessed on a point scale:

- 0 points - clouding of the cornea in the area of the burn is absent;

- 1 point - corneal clouding in the form of a "cloud" (limited gray corneal clouding, barely perceptible during examination);

- 2 points - clouding of the cornea in the form of a "spot" (persistent limited more intense clouding of the cornea in the area of the corneal burn;

- 3 points - the formation of a walleye (persistent clouding of the cornea due to its cicatricial change).

Statistical analysis was performed using the Statistica 7.0 program. For all quantitative data, the group arithmetic mean (M) and standard error of the mean (m) were calculated. The significance of differences between data groups was assessed using Student's t-test and Mann-Whitney U-test, depending on the number of observations and the nature of the distribution of parameter values in the group. Differences were considered significant at p≤0.05 (Table 1).

Table 1

The dynamics of the development of the inflammatory reaction after an alkaline burn of the cornea against the background of the use of lactoferrin solution

No. gr Lactoferrin concentration, mg/ml The severity of the inflammatory reaction in points, days after alkaline burn one 2 3 4 five 6 7 8 nine 10 one Control (0.9% sodium chloride solution) 2.7±0.2 2.5±0.2 2.2±0.2 1.8±0.2 1.8±0.2 1.5±0.2 1.3±0.3 1.2±0.4 0.8±0.4 0.7±0.3 2 0.5 2.5±0.2 2.0±0.1 1.3±0.2* 0.8±0.3* 0.7±0.2* 0.7±0.2* 0.2±0.1* 0* 0* 0* 3 2.5 1.2±0.2* 1.2±0.2* 0.8±0.2* 0.5±0.2* 0.2±0.1* 0* 0* 0* 0* 0* 4 5.0 2.3±0.2 2.0±0.1 1.2±0.2* 1.5±0.2 1.3±0.3 0.8±0.3* 0.7±0.3 0.5±0.2* 0.2±0.1* 0.2±0.1*

* - differences with control are significant, p≤0.05

As can be seen from the data presented in Table 1, in rabbits that, after an alkaline burn, were instilled with a preparation based on lactoferrin at concentrations of 0.5 mg/ml, 2.5 mg/ml and 5.0 mg/ml (5 times a day within 10 days), the degree of inflammatory response was significantly lower than in animals in the control group. The most pronounced relief of inflammation was noted in the experimental group No. 3, in which the animals were instilled with a drug based on lactoferrin at a concentration of 2.5 mg/ml. In these rabbits, by the 6th day of observation, the signs of the inflammatory reaction completely disappeared (Table 1).

Table 2 shows the dynamics of corneal epithelialization in the experimental and control groups after an alkaline burn.

table 2

Dynamics of corneal epithelialization in experimental and control groups after alkali burn

No. gr Lactoferrin concentration, mg/ml The size of the epithelial defect of the cornea in mm ² , days after an alkaline burn one 2 3 4 five 6 7 8 nine 10 one Control (0.9% sodium chloride solution) 42.7±5.0 44.8±4.0 43.3±2.0 36.7±5.0 29.2±6.2 19.7±3.0 7.8±2.5 6.0±2.3 4.0±2.1 2.3±1.5 2 0.5 43.3±2.7 28.7±5.3* 13.7±4.7* 5.7±2.3* 3.0±1.2* 1.5±0.8* 0.8±0.7* 0.3±0.2* 0.3±0.2* 0.3±0.2* 3 2.5 35.3±3.6 14.5±2.8* 7.0±2.8* 2.0±1.0* 0.8±0.7* 0.7±0.6* 0.2±0.1* 0.2±0.1* 0.2±0.1* 0.2±0.1* 4 5.0 45.8±3.3 27.3±7.7* 8.7±3.0* 5.8±3.2* 6.0±3.8* 2.2±1.5* 2.2±1.5* 0.8±0.7* 0.7±0.6* 0.2±0.1*

* - differences with control are significant, р≤0.05

As can be seen from the data, in rabbits in the experimental groups, which were instilled with a drug based on lactoferrin, the process of corneal defect epithelialization proceeded significantly faster than in rabbits in the control group. According to this evaluation criterion, the best therapeutic effect was also achieved when using a drug based on lactoferrin at a concentration of 2.5 mg/ml. So, on the 2nd day of treatment in rabbits, a decrease in the epithelial defect of the cornea was observed by 59%, and on the 10th day of treatment - 99%. While in animals of the control group on the 2nd day of observation, an increase in the epithelial defect by 5% was noted, and on the 10th day - a decrease in the epithelial defect by 95%.

The dynamics of the degree of intensity of corneal opacification in the burn area is presented in Table 3.

Table 3

Dynamics of the degree of intensity of corneal opacity in the burn area

No. gr Lactoferrin concentration, mg/ml severity of corneal opacification in points, day after alkaline burn one 2 3 4 five 6 7 8 nine 10 one Control (0.9% sodium chloride solution) 2.0±0.1 2.0±0.1 2.0±0.1 2.0±0.1 2.0±0.1 2.0±0.1 2.0±0.1 2.0±0.1 2.0±0.1 2.0±0.1 2 0.5 2.0±0.2 2.0±0.2 1.8±0.2 1.8±0.2 1.7±0.1 1.5±0.1* 1.3±0.1* 1.2±0.1* 1.2±0.1* 1.2±0.1* 3 2.5 2.2±0.2 1.2±0.1* 1.3±0.1* 1.3±0.1* 1.3±0.1* 1.3±0.1* 1.2±0.1* 1.2±0.1* 1.2±0.1* 1.2±0.1* 4 5.0 2.0±0.2 2.0±0.2 2.0±0.2 1.7±0.2 1.7±0.2 1.7±0.2 1.5±0.1* 1.5±0.1* 1.5±0.1* 1.5±0.1*

* - differences with control are significant, р≤0.05

In rabbits in the control group, the degree of intensity of corneal opacity equal to 2 points was observed during the entire observation period (11 days after alkaline burn). In rabbits in the experimental groups, which underwent instillation of the drug based on lactoferrin at concentrations equal to 0.5 mg/ml, 2.5 mg/ml and 5.0 mg/ml, a slight decrease in the intensity of corneal opacity in the burn zone was observed. There were no significant differences in the intensity of corneal opacity depending on the concentration of the lactoferrin solution (Table 3).

Thus, evaluating the therapeutic efficacy of lactoferrin administration in relation to the clinical manifestations of burn disease of the cornea of the eye, it should be noted a significant decrease in the severity of the inflammatory response to burn injury, acceleration of epithelium regeneration, a decrease in the degree of clouding intensity and the size of the affected area at all studied concentrations of the drug. The best therapeutic effect was achieved when using a solution of lactoferrin at a concentration of 2.5 mg/ml.

On the 11th day after the burn, the animals were subjected to euthanasia. Histological preparations were examined in a light microscope of the MS 300 series by Micros (Austria) at magnifications: 100, 400, 1000.

The results of histological examination.

Histological examination of the eyes of rabbits in the control group showed detachment (low) and thinning (only 2 layers of cells) of the epithelial layer. An accumulation of exudate was found under the epithelium and loosening of the collagen fibers of the corneal stroma (signs of edema) was noted.

Histological examination of the eyes of rabbits of the second group revealed areas of detachment of the epithelial layer of the cornea from the anterior membrane and stroma. At the same time, the epithelium was very thin (consisted of 1-2 rows of basal cells) and weakly stained. Signs of regeneration were mild or absent. In the stroma, a somewhat loose arrangement of collagen fibers with rounded slits (signs of edema) was observed.

Histological examination of the eyeballs of rabbits of the third group did not disturb the epithelial layer of the cornea. A wider area of hypertrophied epithelial cells was revealed, consisting of 5-6 layers. A somewhat loose arrangement of collagen fibers was observed in the stroma (signs of mild edema).

Histological examination of the eyes of rabbits of the fourth group in the burn area showed detachment (low) and thinning of the epithelium (only 1-2 layers of cells were observed) of the cornea. The epithelial cells were weakly stained. A loose arrangement of collagen fibers (signs of edema) was observed in the stroma.

Thus, in a histological examination of the eyeballs of control rabbits with moderate chemical burns, a morphological picture of exudative and destructive (alternative) inflammatory changes in the corneal tissues was observed in the form of detachment of the stratified squamous non-keratinized epithelium, its thinning and weakening of the ability to stain with hematoxylin and eosin, corneal edema . In these rabbits, the signs of regeneration of the corneal epithelium were weakly expressed and not in all animals.

Histological examination of the eyeballs of rabbits of the second group found that in these rabbits, the histological signs of inflammatory changes in the cornea did not differ from the inflammatory changes detected in animals of the control group. There were inflammatory changes corresponding to the exudative stage of inhibition.

Histological examination of the eyeballs of rabbits of the third group showed inhibition of the exudative stage of corneal inflammation. All rabbits showed signs of corneal regeneration.

Histological examination of the eyeballs of rabbits of the fourth group showed inhibition of the exudative stage of inflammation. However, in these rabbits, as well as in the control group with alkali burns, signs of regeneration of the cornea of the eyes were less pronounced.

Thus, the most pronounced therapeutic effect of the introduction of lactoferrin in relation to an alkaline burn of the cornea of the eye of moderate severity was observed when using lactoferrin at a concentration of 2.5 mg / ml. This concentration was used for further studies.

Example 2 - Experimental modeling of mild acid burn of the central optical zone of the cornea.

Research on male rabbits of the Chinchilla breed (weight 3.0-3.5 kg). Experimental modeling of mild acid burns of the central optical zone of the cornea was performed on 6 rabbits (12 eyes) weighing 3.0-3.5 kg. Acid burn was caused by application of filter paper in the form of a circle with a diameter of 6 mm, moistened with a 3% acetic acid solution with an exposure of 15 seconds to the cornea under local anesthesia with 0.4% inocaine. After removing the filter paper, the eyes were washed with 0.9% sodium chloride solution. Experimental animals were divided into two groups: experimental and control.

- group 1: control group (3 rabbits; 6 eyes). The animals were instilled with isotonic (0.9%) sodium chloride solution (1 drop 3 times a day for 14 days);

- group No. 2: experimental group (3 rabbits; 6 eyes). The animals were instilled with lactoferrin at a concentration of 2.5 mg/ml, 1 drop 3 times a day for 14 days;

Immediately after the application of the burn, the cornea at the site of application of the filter paper became more edematous, initially it was not stained with fluorescein (Figure 1).

In the first group (acid burn, instillations of isotonic (0.9%) sodium chloride solution). The animals showed a pronounced corneal syndrome from 1 to 4 days. Starting from day 1 after the application of corneal burns, a defect in the corneal epithelium appeared within the burnt tissue, the gradual epithelization of which was completed by day 7 (Fig. 2-5). At the end (on day 14), the cornea of all animals was completely transparent and did not differ from intact animals (Fig. 6).

The second group (lactoferrin instillations at a concentration of 2.5 mg/ml (1 drop 3 times a day for 14 days). Severe corneal syndrome was observed during the first day. From 1 to 3 days after corneal burns, a mild swelling of the corneal epithelium, the cornea at the site of the burn was stained superficially only for 1 day (Fig. 7, 8), by 5 days in all animals of the experimental group, the transparency of the cornea was completely restored (Fig. 9). animals, the cornea was completely transparent, did not differ from intact animals.

Example 3 - Experimental modeling of a severe thermal burn of the central optical zone of the cornea.

Research on male rabbits of the Chinchilla breed (weight 3.0-3.5 kg). Experimental modeling of a severe thermal burn of the central optical zone of the cornea was performed on 12 rabbits (24 eyes) weighing 3.0-3.5 kg. A thermal burn was caused by placing a stainless steel cylinder with a base diameter of 6 mm, heated to 200 degrees Celsius, with an exposure of 3 seconds to the central zone of the cornea under local anesthesia with 0.4% inocaine. After applying the burn, the eyes were washed with 0.9% sodium chloride solution. A local anesthetic was re-instilled, followed by scarification of the damaged corneal epithelium. The state of the cornea immediately after the burn is shown in Fig. 10. Experimental animals were divided into two groups: experimental and control.

- group 1: control group (6 rabbits; 12 eyes). Animals were instilled with isotonic (0.9%) sodium chloride solution (1 drop 3 times a day for 28 days);

- group 2: experimental group (6 rabbits; 12 eyes). The animals were instilled with lactoferrin at a concentration of 2.5 mg/ml, 1 drop 3 times a day for 28 days;

In the first group (thermal burn, instillation of isotonic (0.9%) sodium chloride solution (1 drop 3 times a day for 28 days), from 1 to 5 days after corneal burns, within the burnt tissue, the cornea became more edematous , the site of the burn was totally stained with 1% fluorescein solution (Fig. 11, 12).Infiltration of the entire cornea around the burn was observed, which gradually decreased from the periphery to the center, the transparency of the cornea outside the burn was restored from 3 to 7 days (Fig. 12-14) From days 1 to 3, a mixed injection of the bulbar conjunctiva developed, which lasted up to days 7-10. Complete epithelialization of the defect in the first group was observed from days 7 to 13, on average at 9.2 ± 2.1 days. eye on the fourth day (8.33%). burn (Fig. 15).

The second group: thermal burn, treatment with lactoferrin. From 1 to 4 days after the application of corneal burns, within the burnt tissue, the cornea became more edematous, the burn site was totally stained with 1% fluorescein solution. Infiltration of the entire cornea around the burn was observed, which gradually decreased, the transparency of the cornea outside the burn was restored from 2 to 4 days (Fig. 16-17). During the first day, a mixed injection of the bulbar conjunctiva developed, which lasted up to 5-7 days (Fig. 18). Complete epithelization of the defect in the first group was observed from 6 to 8 days, on average - at 6.8±0.75 days (Fig. 19). No perforations were observed. The size formed to 28 days of turbidity, on average, amounted to 27±12.1% of the diameter of the cylinder, which caused the burn (Fig.20).

Thus, instillations into the conjunctival cavity of an aqueous solution of lactoferrin 2.5 mg / ml, 1 drop 3 times a day for twenty-eight days after a severe corneal burn, help to reduce the intensity of the inflammatory reaction, reduce the area of the corneal defect, accelerate its healing, and also form less coarse and smaller in diameter haze. On the background of treatment with a drug based on lactoferrin, no development of perforations is observed.

The use of the invention in clinical practice will allow:

- increase the effectiveness of the treatment of corneal burns by reducing the severity of oxidative stress;

- accelerate the epithelialization of corneal defects;

- reduce the severity of the inflammatory response to burn injury;

- accelerate the process of restoration of defects in the epithelium and stroma of the cornea;

- reduce the intensity and area of corneal opacity.

Claims (4)

A method for treating corneal burns, including instillation of an antioxidant drug into the conjunctival cavity, characterized in that an aqueous solution of lactoferrin 2.5 mg/ml is used as an antioxidant drug according to the following scheme: - with alkaline burns - 2 drops 5 times a day for 10 days; - with acid burn - 1 drop 3 times a day for 14 days; - with thermal burns - 1 drop 3 times a day for 28 days.

Images