RU2818909C1 - Method for brain neuroprotection in simulating photoinduced ischemic stroke - Google Patents

Abstract

FIELD: experimental medicine; neurology.

SUBSTANCE: rats with photoinduced ischemic stroke are subject to 2 hour inhalation with krypton-oxygen mixture of Kr 70%/O₂ 30% at 0.5 l/min per one animal.

EFFECT: method enables determining the clinical effectiveness and safety of using the inert gas krypton as a neuroprotector in focal cerebral ischemia.

1 cl, 2 dwg, 1 ex

Description

The invention relates to medicine, and can be used to determine the clinical effectiveness and safety of the use of the inert gas krypton as a neuroprotector in focal cerebral ischemia (CB).

State of the art.

To date, there are several examples of the use of gas mixtures containing krypton in the mode of inhalation effects on the body for various purposes.

In particular, the use of krypton in the treatment of somatoform mental disorders is known, including inhalation of an inhaled mixture of krypton and oxygen in a ratio of 50/50 to 80/20 vol. % through an open or closed breathing circuit using an inhalation device controlled by a gas analyzer (RU 2769783). However, this method of exposure does not allow us to draw conclusions about the degree of influence of inhalation exposure to krypton on the state of the brain affected by ischemic stroke.

There is also a known method for treating pain during the rehabilitation of cancer patients after spinal surgery, which involves inhaling a mixture of krypton and oxygen in a ratio of 70/30 vol. %. For 5-7 minutes in one session (RU 2713455). The technical result of the known method is to reduce pain with minimal side effects by reducing the dose of opioid analgesics. This method of exposure, like the previous one, does not allow us to draw conclusions about the degree of influence of inhalation exposure to krypton on the state of the brain affected by ischemic stroke.

It is also known to use a gaseous composition containing krypton as an inhaled drug for the prevention or treatment of neurointoxication in humans, in which gaseous krypton is the active principle and the volume fraction of krypton is from 15 to 80% (WO 2012025677). As indicated in the description of the known method, the technical result of its use is the treatment of neurointoxication resulting from or a consequence of traumatic brain injury, cerebrovascular accident of the central nervous system, cerebral ischemia, taking an addictive substance, Alzheimer's disease, as well as Parkinson's disease, Huntington's chorea, amyotrophic lateral sclerosis, acute disseminated encephalomyelitis, tardive dyskinesia, olivopontocerebellar atrophy, schizophrenia or epilepsy. Moreover, gaseous krypton is proposed to be used in a very wide range of dosages from 15 to 80%. In this regard, the described method of exposure does not allow us to draw conclusions about the exact concentration and duration of inhalation exposure to krypton in order to level out damage to the brain during ischemic stroke.

It was not possible to find methods for neuroprotection of the brain when modeling photoinduced ischemic stroke in the information database available to the authors.

Disclosure of the invention.

Currently, there is an increased attention of clinical medicine to the effects of inert gases on the human body due to the inability of inert gases to enter into chemical compounds with other elements of the periodic table D.I. Mendeleev, as well as their ability to exert biological effects on a living organism, entering and leaving the body unchanged. Krypton is one of the representatives of inert gases, which has no odor, no color, no radioactive properties, and is several times denser than air. Its biological properties are still poorly studied.

The purpose of this study was to evaluate the effect of a 2-hour inhalation of a krypton-oxygen mixture (Kr 70%/O ₂ 30%) after modeling photoinduced ischemic stroke (PII) on the severity of neurological deficit and the degree of brain damage in rats compared to the control group, in which a 2-hour inhalation of a nitrogen/oxygen gas mixture (N ₂ 70%/O ₂ 30%) was performed

In the FII model, which is based on focal damage to the endothelial lining of the vessels of the rat brain cortex, faster adaptation and organization of this area was discovered under the influence of 2-hour inhalation of a krypton-oxygen mixture, as evidenced by both the results of histological and immunohistochemical studies, as well as clinical data - PKO test.

Neurological examination data based on the results of the PCO test.

On the third day of observation after FII modeling, no significant difference in the total scores of the PCO test was detected in the Krypton and Azot groups (p = 0.4020). On the 7th day of observation, a significant difference was noted in the Krypton group (p = 0.0245), which may indicate a faster rehabilitation of animals in the Krypton group.

Data from morphological studies, such as MRI of the brain, histological and immunohistochemical studies of the brain, leave no doubt that in the Krypton group there is a significant acceleration of the processes of repair and organization of the ischemic focus and penumbra zone. In particular, according to the results of an MRI study, the area of the lesion in the brain was significantly smaller in the Krypton group compared to the Nitrogen group and averaged 17.56 [14.18; 20.38] mm ² and 22.19 [17.98; 24.42] mm ² , respectively (p = 0.0435).

The activation of regeneration processes in the Krypton group is evidenced by a faster, compared to the Nitrogen group, regenerative process, scar formation (proliferation of connective tissue), and vascular formation in the necrosis zone. In the Azot group, there was a tendency towards delimitation of the ischemic focus with the formation of a cell shaft in the periphery and a necrotic focus in the center, which indicates the beginning of the formation of a cystic-glial formation at the site of cerebral infarction. It has been established that the rehabilitation potential is an order of magnitude higher in the Krypton group.

In histological preparations of GM animals of the Krypton group, plethora of the soft membranes of the brain was noted. No thickening of the meninges was detected. Congestion of newly formed vessels in the damaged area was noted. The area of damage was represented by necrotic detritus with a diffuse proliferation of connective tissue in the form of a cellular structure in which microgliocytes were located. Newly formed vessels were found both at the periphery and in the center of the damage zone. Along the periphery of the damaged area, the brain tissue is sparse due to edema; in some specimens, cyst-like formations without epithelial lining were noted. In the penumbra zone, neurons with signs of damage were identified (dark neurons, nuclear displacement, nucleolar displacement, hyperchromic nuclei, hypochromic nuclei, swollen nuclei).

In the Nitrogen group, soft membranes were present on most preparations. The soft membranes are partially exfoliated and thickened in all specimens. The vessels were full of blood, some were hyalinized. The area of damage in the cortex, the central part of which, in the Nitrogen group is represented by a homogeneously stained area of necrotic detritus, surrounded by a shaft of glial cells, macrophages with granular cytoplasm, foam cells at different stages of apoptosis. Full-blooded capillaries were observed in the cell shaft area. In all preparations from the Nitrogen group, foam cells, glial cells and newly formed vessels were identified at the periphery of the damaged area. In the penumbra zone, neurons with signs of damage were noted (dark neurons, nuclear displacement, nucleolar displacement, hyperchromic nuclei, hypochromic nuclei, swollen nuclei)

The observed diffuse proliferation of connective tissue, uniform distribution of microgliocytes in the damaged area, the presence of vessels both in the center and along the periphery of the damaged area indicate more pronounced regeneration on preparations of the Krypton group, compared to preparations from the Nitrogen group, on which no growth of connective tissue was observed , microgliocytes formed a shaft along the periphery of the area of necrotic detritus, and foam cells at different stages of alteration were also observed in this zone.

As a result of an immunohistochemical study, it was established that immunohistochemical staining NeuN, which reveals a nuclear protein characteristic only of neurons, in histological preparations of both groups revealed neurons in the area of necrosis of the cerebral cortex. NeuN positive cells were also detected in the center of the damage zone. No neurons were found in the organization zone. Outside the damage, the nuclei and cytoplasm of neurons are intensely stained.

Immunohistochemical detection of microglial markers in Iba in the Azot group revealed a small number of microglial cells localized in the necrosis zone. Few microglial cells were also found in the area of necrosis. In the Krypton group, a large number of diffusely distributed iba-positive cells were identified, both in the zone of necrosis and in the zone of organization of necrosis.

Immunohistochemical detection of GFAP, which is a marker of astrocytes, showed that GFAP-positive cells are located on the outside of the necrosis zone at the border with the brain tissue in both groups.

Thus, as a result of the study, it was established that 2-hour inhalation of a krypton-oxygen mixture Kr 70%/O ₂ 30% has a neuroprotective effect in FII, which is expressed in the positive dynamics of the neurological status (PCO test “Placing the limb on support”) on 7th day of observation, a decrease in the focus of ischemia according to MRI studies, as well as more pronounced processes of repair and regeneration according to histological and immunohistochemical studies.

Implementation of the invention.

Experiments were carried out on male Wistar rats weighing 250-300 g (n=20). On the eve of the experiment, the animals did not receive food for 8 hours, but had free access to water. The study protocol was approved by the Local Ethics Committee of the Federal Scientific and Clinical Center RR 3/22/3 dated December 14, 2022. The experiments were carried out in accordance with the requirements of Directive 2010/63/EU of the European Parliament and the Council of the European Union for the Protection of Animals Used for Scientific Purposes.

The animals were randomly divided into 2 groups depending on the volume of interventions performed:

- control group with FII + inhalation N ₂ 70%/O ₂ 30% (Nitrogen group), n=10;

- experimental group with FII + inhalation Kg 70%/O ₂ 30% (Krypton group), n=10.

FII modeling.

After intraperitoneal administration of 6% chloral hydrate at a dose of 300 mg/kg, FII modeling was performed in accordance with the well-known methodology [Silachev D.N., Uchevatkin A.A., Pirogov Y.A., Zorov D.B., Isaev N.K. Comparative evaluation of two methods for studies of experimental focal ischemia: magnetic resonance tomography and triphenyltetrazoleum detection of brain injuries. Bull Exp Biol Med. 2009; 147 (2): 269-72. doi: 10.1007/s10517-009-0489-z. PMID: 19513437]. FII was modeled in the sensorimotor cortex of the brain rats using photochemically induced thrombosis of the vessels of the brain cortex. Photosensitive dye rose Bengal (3%, 40 mg/kg intravenously; Sigma-Aldrich, St. Louis, MO, USA) was injected into the jugular vein. After this, the rat's head was fixed in a stereotaxic frame (Bregma stereotaxic coordinates: 0.5 mm distally and 2.5 mm laterally), the skull was exposed through a midline incision, and the periosteum was cleared. Next, the brain hemisphere in the area of the sensorimotor cortex was irradiated with green light at λ=550 nm for 15 minutes. After suturing the skin, the rats were placed in a cage under an infrared heat lamp until they recovered from anesthesia. Body temperature was maintained at 37±0.5°C throughout the experiment. Thermometry was performed by installing a rectal body temperature sensor, and thermoregulation was carried out automatically by connecting the heating module to a thermostat and setting limit values.

Exposure to krypton.

After awakening, the animals were placed in a transparent plastic chamber with a volume of 36 liters, into which a fresh gas mixture (N ₂ 70%/O ₂ 30% - Nitrogen group; Kr 70%/O ₂ 30% - Krypton group) was constantly supplied with a flow of 0.5 l/min per animal. No more than 5 animals of the same group were in the chamber at one time, which made it possible to avoid hypoxia and hypercapnia.

The exposure time in the chamber was 2 hours. During the entire experiment, continuous monitoring of the level of O ₂ and CO ₂ in the chamber with animals was carried out using an indoor atmosphere control unit (ZAO INSOVT St. Petersburg, Russia). After the end of the exposure period, the animal’s general condition (level of wakefulness, mobility) and pain relief (paracetamol 50 mg/kg s.c.) were assessed. Then the animals were moved to a cage with free access to water and food.

On the 3rd day (D3), 7 days (D7) and 14 days (D14) after the stroke, the following assessment studies of the condition of the experimental animals were carried out.

The neurological status of the animals was assessed using the PCO test. A protocol was used based on the technique described by De Ryck et al. (1989) [De Ryck M., Van Reempts J., Borgers M., Wauquier A., Janssen P.A. Photochemical stroke model: flunarizine prevents sensorimotor deficits after neocortical infarcts in rats. Stroke. 1989; 20 (10):1383-90. doi: 10.1161/01.str.20.10.1383. PMID: 2799870] and modified by J. Eolkkonen et al. (2000) [Jolkkonen J., Puurunen K., Rantakomi S., Harkonen A., Haapalinna A., Sivenius J. Behavioral effects of the alpha(2)-adrenoceptor antagonist , atipamezole, after focal cerebral ischemia in rats. Eur J Pharmacol. 2000; 400 (2-3): 211-9. doi: 10.1016/s0014-2999(00)00409-x. PMID: 10988336]. Rats were hand habituated for a week before testing. The test consisted of seven trials assessing sensorimotor integration of the forelimbs and hindlimbs in response to tactile, proprioceptive, and visual stimulation. Each test was scored as follows: the rat performed the test normally - 2 points; the rat completed the test with a delay of >2 s and/or not completely - 1 point; the rat did not complete the test - 0 points.

In FIG. Figure 1 shows the dynamics of changes in PQS results in both groups in the form of the sum of points for the test.

MRI study.

On the 14th day after the stroke, an MRI study of the animals was carried out on a tomograph with a magnetic field induction of 7 T and a gradient system of 105 mT/m (BioSpec 70/30, Bruker, Germany). Anesthesia was performed with isoflurane (1.5-2%), after which the rat was placed in a positioning device with a stereotaxis and thermoregulation system [Silachev D.N., Uchevatkin A.A., Pirogov Y.A., Zorov D.B., Isaev N.K. Comparative evaluation of two methods for studies of experimental focal ischemia: magnetic resonance tomography and triphenyltetrazoleum detection of brain injuries. Bull Exp Biol Med. 2009; 147 (2): 269-72. doi: 10.1007/s10517-009-0489-z. PMID: 195134370 error! Reference source not found.].

A standard protocol for studying the rat brain was used, which includes obtaining T2-weighted images. A linear transmitter with an internal diameter of 72 mm was used to transmit the radio frequency (RF) signal, and a rat brain surface receiving coil was used to detect the RF signal. The following pulse sequences (PS) were used: RARE - spin echo-based TP with parameters: TR = 6000 ms, TE = 63.9 ms, slice thickness 0.8 mm with a step of 0.8 mm, matrix size 256 × 384, resolution 0.164 × 0.164 mm/pixel. The total scanning time for one animal was about 25 minutes. The degree of damage to the brain was assessed using graphical analysis of MRI images with calculation of the volume of the damaged area of the brain. To do this, a slide with the largest area of brain damage was selected from a series of MRI images. Using the ImageJ program (National Institutes of Health image software, Bethesda, MD, USA), the damage area in ^mm2 was calculated. Next, the area of damage to the brain was calculated in a similar manner on four more slides (two cranial and two caudal). The volume of damage to the brain was calculated using the following formula: V = ΣSn × d, where d is the thickness of one slice (0.8 mm), ΣSn is the sum of the damage areas on five slides (mm ² ). Mortality in animal groups was assessed after 24 hours, on the 7th and 14th days after the stroke.

Figure 2 shows the volume of the damage zone on the 14th day of observation according to the results of an MRI study of animals.

Histological examination.

Histological examination (determining the volume of the lesion) in rats was carried out on the 14th day after TBI immediately after euthanasia (decapitation under anesthesia with 6% chloral hydrate). Brain pieces were fixed in 4% buffered formalin for 24 hours. Further studies were carried out using standard paraffin wiring. The resulting frontal sections, 4 µm thick, were stained with hematoxylin and eosin using Nissl staining. Morphometry was carried out after obtaining digital images on a Nikon Eclipse Ni-U microscope (Japan) lenses: 4x, 10x, 20x 40x. DS-RI2 camera. Morphometry was carried out using the NIS-Elements BR “Nikon” program (Japan). Morphological changes in the infarct area and penumbra, as well as the area of the damaged area, were assessed.

Immunohistochemical study

Paraffin blocks of deceased rats diagnosed with non-traumatic subarachnoid hemorrhage and hemorrhagic stroke in the amount of 56 cases were selected as the study group; the control group consisted of 7 cases of deceased rats diagnosed with acute coronary death. Exclusion criteria were: trauma, malignant diseases. Material provided by the Moscow Bureau of Forensic Medicine. Subsequently, the blocks were cut into 5-μm sections on a microtome microtome and stained. Hematoxylin and eosin and Nissl stains were used. For immunohistochemical studies, stains were used to detect Iba and NeuN antigens. Sections were dewaxed in xylene and dehydrogenated in ethanol. High-temperature unmasking was carried out in citrate buffer pH 6 (Target Retrieval Solution, DAKO, Glostrup, Denmark). Sections were cooled and washed three times in distilled water and three times in phosphate buffered saline (PBS) for 5 minutes each (PBS ShS Wash Buffer + Tween, Cell Marque, Rocklin, CA, USA). To suppress endogenous peroxidase, sections were kept in 3% hydrogen peroxide for 10 minutes. To prevent nonspecific binding of primary or secondary antibodies to tissue proteins, (Protein Block Serum-free, Abeam, Cambridge, UK) was used with an exposure time of 15 minutes. Sections were incubated at 37°C for 1 hour with primary antibodies (ab5076, 1:500, ab68428 1:500, and ab177487 1:200 dilution in Antibody Diluent ab64211, Abeam, Cambridge, UK). The sections were then washed in PBS twice for 5 minutes each. The intensity of the reaction was determined using a detection kit (ab64264, Abeam, Cambridge, UK). After washing the sections in PBS, they were counterstained with hematoxylin. After washing in running water, the sections were dehydrated and mounted under a coverslip. Analysis was performed on a Nikon Eclipse Ni-e microscope using NIS-Elements and ImageJ software.

Statistical analysis.

Statistical data processing was carried out using STATISTICA 10.0 (StatSoft. Inc., USA) and GraphPad Prizm programs. The normality of the distribution of the trait in the samples was assessed using the Shapiro-Wilk test. All data are presented as median (interquartile range). Statistical differences in data that had a non-normal distribution in at least one of the groups were analyzed using the Mann-Whitney U test with a Bonferroni correction for comparisons of three or more groups, as well as the Kruskal-Wallis test or the Mann U test. Whitney for analysis of no more than 2 groups. The criterion for statistical significance was the level of P<0.05.

Thus, it has been reliably established that the inert gas krypton has biological activity against focal ischemic damage to the brain.

Claims (1)

A method of neuroprotection of the brain in vivo, which consists in the fact that rats with photo-induced ischemic stroke are given a 2-hour inhalation of a krypton-oxygen mixture Kr 70%/O ₂ 30% with a flow of 0.5 l/min per animal.