RU2258521C1 - Biotransplant and method for treating ischemic insult - Google Patents

Abstract

FIELD: biopharmacology, medicine.

SUBSTANCE: the suggested biotransplant contains an active component: the culture of genetically unmodified neuronal stem cells (NSC) obtained out of anterior cerebral tissue of human embryos of the 1^st trimester of pregnancy or periventricular cerebral area of 15-20 wk gestation and selectively multiplied under cultivating conditions up to the quantity of 10 (7) - 10 (9) pluripotent undifferentiated cells in composition of neurospheres, at the content of NSC being 50-500 mln., and 2 ml physiological solution. The method for treating ischemic insult deals with introduction of 50-500 mln. NSC in 2 ml physiological solution. The suggested biotransplant and method for treating ischemic insult enable to quickly restore and improve cerebral functions.

EFFECT: shortened terms of therapy.

5 cl, 2 ex, 1 tbl

Description

The invention relates to a culture of genetically unmodified neuronal stem cells and a method for the treatment of ischemic stroke.

Acute cerebrovascular accident (stroke) is an urgent medical and social problem of modern medicine, due to their high share in the structure of morbidity and mortality, significant indicators of temporary labor losses and primary disability. Mortality from vascular diseases of the brain in our country, among which ischemic and hemorrhagic strokes are especially distinguished, ranks second in the structure of total mortality, not much inferior to mortality from heart diseases. Mortality in the acute stage of stroke is 35%, increasing by 12-15% by the end of the first year. Disability due to stroke ranks first among all causes of primary disability. The consequences of a stroke are expressed in damage and often the death of a part of the brain affected by a stroke that can no longer perform its functions, which entails all kinds of impaired speech, consciousness, coordination of movements, vision, sensitivity and paralysis.

Existing methods of treatment using cerebro- and angioprotectors, nootropics and biogenic stimulants are not effective enough and allow to achieve only some weakening of clinical manifestations.

Currently, thanks to advances in neurobiology, neurotransplantation is used to treat various brain injuries.

The main limiting factors in the use of neural tissue for transplantation are the age of the donor (Ferry, 1989. Dunnett, 1991), a method for preparing a biograft and a method for administering a biograft,

- neurosurgical intracerebral transplantation of a suspension of immature neural tissue cells or stem nerve cells,

- subcutaneous administration of a suspension of immature nervous tissue cells.

With intracerebral (intracerebral) transplantation, complications such as perifocal cerebral edema, as well as microbleeds in the area of the operation are possible, which can cause additional complications and, as a consequence, a slower and slightly pronounced restoration of lost functions.

Endolumbar transplantation is much more effective than subcutaneous injection, and has several advantages compared to neurosurgical stereotactic intracerebral transplantation.

It is known from liquorology that cerebrospinal fluid washing the spinal cord tends upward to the brain, and cerebrospinal fluid flows downward, at the level of the medulla oblongata, they mix and penetrate in both directions. This suggests that

transplanted cells introduced endolumbally through cerebrospinal fluid can penetrate the trunk, cortex, ventricles of the brain, and then migrate to the lesion and directionally differentiate into neurons, astro- and oligodendroglia;

cerebrospinal fluid (subarachnoid) space is an adequate place for transplanted cells due to the sufficient delivery of oxygen and nutrients, as well as low tissue pressure on the graft (5);

the effect of endolumbal introduction of cells on brain processes may be due to the fact that neurotrophic factors containing endorphins, enkephalins, nerve growth factor and many other neuropeptides that promote the growth and regeneration of nerve elements are secreted by transplanting transplanted cells and can be transferred with blood flow, reaching the focus damage;

with endolumbal transplantation, brain tissue, meninges and blood vessels of the brain are not damaged, which significantly reduces the possibility of post-transplant complications.

The effect of the operation can be significantly improved by increasing the amount of transplanted material, as well as by pretreating the transplanted neurons with growth (neurotrophic) factors to increase survival and stimulate their differentiation. The discovery of the role of neuronal growth factors in suppressing neurodegenerative processes (Tuszynski M.H. et al., 1998) suggests the use of growth factors for the treatment of neurodegenerative diseases.

The known method of treating ischemic stroke, selected as a prototype, has disadvantages: a slower and slightly pronounced restoration of lost functions, longer treatment periods and repeated repetition of courses.

The present invention is the creation of a biograft containing neuronal stem cells (NSCs) for endolumbar transplantation, which eliminated side effects and increased the effectiveness of the treatment of ischemic stroke.

To solve this problem, a group of inventions is proposed, united by a common inventive concept.

As a biograft use the laboratory line of human pluripotent NSC (patent No. 2191388 from 12/18/2002). A culture of genetically unmodified NSCs obtained from the forebrain tissue of first-trimester human embryos or the periventricular region of the brain of fetuses of 15-20 weeks of gestation is a suspension of NSC clones selectively propagated under cultivation to an amount of 10 (7) -10 (9 ) pluripotent undifferentiated cells in the composition of the neurospheres.

A method for endolumbar administration of ischemic stroke from 50 to 500 million neuronal stem cells is proposed. The introduction is carried out in 2-5 ml of physiological saline solution simultaneously. In cases of insufficient effectiveness, the procedure is repeated after 6-12 months to improve motor, cognitive, sensitive disorders.

The method of treatment is based on the fact of the substitution effect of neuronal stem cells experimentally proved by the authors. The biological product also has a pronounced stimulating effect on brain function. A biological preparation also improves the immunological status of patients.

The method is performed as follows:

Previously, one hour before neuronal stem cell transplantation, a patient with an ischemic stroke in the acute phase is injected intramuscularly with 12 mg of an official dexazone solution for immunosuppressive purposes.

Next, a patient with ischemic stroke, in a small operating room, subject to all the rules of aseptic and antiseptic in the supine position, is treated with 70 degrees alcohol. After that, in the interval between the third and fourth lumbar vertebrae, a disposable sterile needle for lumbar puncture is punctured until the cerebrospinal fluid appears. After that, the cerebrospinal fluid pressure is measured and 5 ml of cerebrospinal fluid is taken for general analysis. Then, through the same needle, 150 million sterile living neuronal cells are injected in two ml of physiological saline once, slowly for two minutes. Then the needle is removed and an aseptic dressing is applied. The patient is assigned bed rest during the day. On the second or third day, an improvement in the patient's condition is observed in the form of a decrease in the degree of impaired consciousness, an improvement in motor and cognitive functions. Neurological status was assessed, psychological tests were conducted, and the bioelectrical activity of the brain was evaluated according to EEG data.

After 1.5 months, the patients experienced a second phase of improvement in the form of improved motor, cognitive functions, and a decrease in sensitivity disorders.

Specific examples of the application of the proposed invention.

Example 1

Patient K., 40 years old. Diagnosis: Ischemic stroke in the vertebral-basilar arterial system from 03/28/03. Coronary heart disease. Hypertension III Art.

On admission, he complained of headache, hiccups, numbness in the right half of the face, double vision, weakness in the right limbs. The above complaints developed acutely on the day of admission against the background of a rise in blood pressure to 220-110 mm Hg. Neurological status: in consciousness, there are no meningeal signs. Pupils S> D, photoreaction of the pupils saved. Horizontal nystagmus with abduction of eyeballs to the left. Right-sided moderate ptosis, the right nasolabial fold is smoothed, the tongue is in the midline.

The picture of right-sided moderate hemiparesis according to the central type is up to three points. Tendon reflexes D> S. Left-sided hemigipesthesia. Coordination tests are performed uncertainly on the right.

Within two weeks of being in the intensive neurology unit, the patient received vascular, metabolic, hypotensive, disaggregant, neuroprotective therapy. It was possible to achieve stabilization of hemodynamic parameters, but no clear improvement in neurological status was noted.

On the 14th day of hospital stay, the patient underwent endolumbar transplantation of neuronal living stem cells, the manipulation was satisfactory.

The first positive effect was observed during the first three days after the manipulation, a repeated positive effect after 1.5 months. The patient's degree of paresis decreased from 3 to 1 point, sensitivity disorders on the face and limbs regressed, diplopia, nystagmus disappeared. The psychoemotional state is satisfactory.

Example 2

Patient B., 49 years old.

Diagnosis: Ischemic stroke in the pool of the right middle cerebral artery from 3.11.2003, ischemic heart disease. Hypertension III. Angina pectoris II functional class.

Upon admission, she complained of a diffuse headache, the inability to move her left limbs.

In neurological status: Level of consciousness - stunning. At times disoriented, inadequate. Meningic signs are not revealed. Pupils D = S, photoreaction saved. The movement of the eyeballs in full. Asymmetry of the face, deviation of the tongue due to central lesion of the VII, XII pairs of cranial nerves on the left. The motor sphere: deep central left-side hemiparesis to plegia in the arm and 1 point in the leg. Symptom of Babinsky on the left. Left-sided hemigipesthesia.

During 11 days in the intensive neurology unit, the patient received vascular, metabolic, hypotensive, disaggregant, neuroprotective therapy. It was possible to achieve stabilization of hemodynamic parameters, however, a clear improvement in neurological status was not observed.

On the 11th day after the onset of the disease, the patient underwent endolumbar transplantation of neuronal living stem cells, the manipulation was satisfactory.

The first positive effect was observed during the first three days after the manipulation, a repeated positive effect after 1.5 months. The patient noted a restoration of consciousness to a clear level. Cognitive functions with significant improvement: the patient is adequate, contact, oriented. There was a decrease in the degree of paresis to 1 point in the hand and 3 points in the leg. Significantly regressed the phenomenon of hemihypesthesia. The effects of damage from the cranial nerves decreased: the face is symmetrical, the tongue is a slight deviation. The psychoemotional state is satisfactory.

The effectiveness of the proposed method

The treatment results for the proposed method and prototype are presented in table. 1, where the fact of a more rapid improvement in brain function and a reduction in treatment time was recorded.

In the proposed method achieved: 1. A faster recovery of the degree of impaired consciousness (12% compared with the prototype) 2. Regression of motor impairment (10%) 3. Faster recovery of cognitive functions according to psychological tests. 4. Reducing the treatment of the acute period of stroke by 5-6 days.

Table Ways
treatment

Dynamics Number
sick Positive Treatment Results Impaired consciousness Cognitive function Motor disorders Abs. IN% Abs. IN% Abs. IN% Prototype treatment 72 40 55.6 29th 40.3 39 54,2 The treatment according to the proposed method 31 21 67 16 51.6 twenty 64.5

Claims (5)

1. A biograft for the treatment of ischemic stroke, characterized in that as an active component it contains a culture of genetically unmodified neuronal stem cells (NSC), obtained from the tissue of the forebrain of human embryos of the 1st trimester of pregnancy or the periventricular region of the brain 15-20 weeks of gestation and selectively propagated under cultivation conditions to an amount of 10 (7) - 10 (9) pluripotent undifferentiated cells in the composition of the neurospheres. 2. A method of treating ischemic stroke, characterized in that the patient is endolumbally simultaneously injected with a biograft according to claim 1. 3. The method according to claim 2, where the patient is injected from 50 to 500 million neuronal stem cells. 4. The method according to claim 2, where the patient is previously intramuscularly administered dexazone. 5. The method according to claim 2 for improving motor, cognitive, sensory disorders, the procedure is repeated after 6-12 months.