RU2283121C2 - Method for treating stroke cases - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method involves carrying out intracerebral implantation of spinal marrow stem cells during neurosurgical intervention in the injured area. Transplantation is carried out in early and late recovery period after stroke. The whole autologic mononuclear cells complex is introduced as the stem cells separated from spinal marrow aspirate without selecting individual cell fractions and their culturing.

EFFECT: simplified treatment course; reduced neurological deficiency.

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Description

The invention relates to medicine and can be used in neurology and neurosurgery for the treatment of patients with persistent neurological deficit after an ischemic or hemorrhagic stroke.

It is known that the restoration of nervous tissue after acute cerebrovascular accident (stroke) may be due to the ability of stem cells to differentiate into neurons, as well as their production of soluble factors that stimulate the proliferation of neuronal precursors and protect brain cells from apoptosis.

In an adult, stem cells are localized mainly in the bone marrow. In an in vitro culture, the ability to differentiate bone marrow stem cells in a neuronal direction has been shown. These cells can cross the blood-brain barrier and penetrate into the brain tissue, as a source of regeneration of nerve tissue. With this in mind, it was proposed to use stem cell transplantation as a replacement therapy for injuries and / or diseases of the brain.

The validity of this approach was confirmed in experimental models of brain damage. Thus, in a rat ischemic stroke model, intravenous or intracerebral administration of various types of stem cells was accompanied by restoration of brain function and a decrease in the severity of neurological deficiency 2 weeks after occlusion of the middle cerebral artery [1, 2, 3].

Fetal cells derived from human brain tissue from abortive human embryos are another source of stem cells, including neuronal precursors used in replacement therapy for neurodegenerative diseases and brain injuries. Thus, a method for the treatment of diseases and injuries of the central nervous system (CNS) based on the use of stem nerve cells obtained from the tissue of the forebrain or periventricular region of the brain of abortive human embryos of the first trimester of pregnancy is proposed. In this case, stem cells are selectively isolated from the obtained material, which before use grow up to 10 ⁷ -10 ⁹ pluripotent undifferentiated cells in culture (patent No. 2191388, RU, 2002).

There is also a method of treating central nervous system diseases, according to which 1 ml of a suspension of human brain embryonic tissue is injected into the cerebrospinal fluid of the brain together with protein-nucleic extracts of this tissue in an amount of 0.01-0.05 mg of the dry residue of the mixture per 1 kg of patient weight (patent No. 21429427, RU, 1999).

The use of fetal stem cells, however, has moral and ethical limitations, difficulties in obtaining human abortive embryos, is associated with difficulties in isolating and culturing cells, and is also associated with a high risk of bacterial and / or viral microflora contamination of the material. In addition, the possibility of malignant transformation of embryonic cells is actively discussed in the literature.

Another source of neuronal cells are immortalized cell lines [4]. For example, NTera 2 / cl / Dl (NT2) cells, originally isolated from a testicular carcinoma tumor, can differentiate into neuronal cells (LBS neurons) in response to retinoid acid treatment. In 2000, phase I clinical trials were conducted to assess the safety and tolerance of intracerebral transplantation of such cells in 12 stroke patients with persistent neurological deficit [5]. Transplantation was performed on average 2.2 years after a stroke (from 7 months to 6 years), and cells were administered once at a dose of 2 or 6 × 10 ⁶ / patient. The authors showed good tolerance and safety of transplantation of LBS neurons and a significant decrease in the severity of neurological deficit. When assessed according to the Barthel scale, an increase in the total score was noted by an average of 10%, but only with the introduction of 6 × 10 ⁶ cells / patient. Significant disadvantages of using immortalized cell lines are the need for their cultivation and cryopreservation, the risk of transplanted cells returning to the tumor phenotype, and the need for prolonged (1 week before and 8 weeks after transplantation) immunosuppressive therapy with cyclosporin A to prevent rejection of transplanted allogeneic cells.

The most attractive is the use of stem cells isolated from bone marrow or adipose tissue of an adult. So, there is a method of intracerebral bone marrow hematopoietic stem cell transplantation in the treatment of human neurodegenerative diseases (Alzheimer's, Parkinson's, Kreutzfeldt-Jakob disease) (patent No. 2216336, GB, 2003). There is also known a method of using mesenchymal stem cells (MSCs) isolated from human adipose tissue in models of traumatic and degenerative diseases of the central nervous system in experimental animals (patent No. 793173, USA, 2001).

With the aim of treating stroke, a method is proposed (patent AU 5130000, A 61 K 35/00, 2000), according to which a MSC fraction adhering to plastic is isolated from mononuclear cells of the bone marrow of a person, which are further grown in vitro and then introduced in various ways (intracerebral, intracarotid, intravenous) to laboratory animals (rats and mice) 24 hours after a 2-hour occlusion of the middle cerebral artery (experimental model of acute cerebrovascular accident), and a decrease in neurological severity is observed on days 14-28 Skogen deficit. For a more efficient growth of MSCs and enhance their differentiation in the neuronal direction, a long-term (for 1 month) cultivation of bone marrow stem cells in the presence of a complex of growth factors, including stem cell factor (SCF, 100 ng / ml), nerve growth factor (NGF, 200 ng / ml), a neurotrophic factor isolated from the brain (BDNF, 100 ng / ml) and epidermal growth factor (EGF, 20 ng / ml). The authors of the patent propose to use this approach in the treatment of the consequences of acute cerebrovascular accident in humans. However, clinical studies of the effectiveness of the developed method in the treatment of stroke in humans have not been conducted. It should be noted that the experimental model of stroke in animals is fundamentally different from the real clinical situation, in which the duration of cerebrovascular accident can significantly exceed 2 hours and lead to more pronounced brain damage. Stem cell transplantation 24 hours after the stroke appeared to be problematic due to the severity of the general condition of the patient and the severity of the systemic inflammatory response. In addition, stem cells are introduced into the area of the ischemic brain without taking into account the localization of damage to functionally important brain structures. Another disadvantage of the proposed method is the complexity of the technology of isolation and long-term cultivation of mesenchymal stem cells and, as a result, the high risk of their contamination by pathogenic microflora and the high cost of consumables, including growth factors.

An object of the present invention is to provide a method for the treatment of stroke based on autologous transplantation of bone marrow stem cells obtained by a simplified and more economical technology, which allows to reduce the severity of neurological deficiency in patients in the early or long-term period after an ischemic or hemorrhagic stroke, without causing any Any adverse or toxic reactions.

The problem is solved by using a fraction of autologous mononuclear cells isolated from a patient’s bone marrow aspirate, which, after determining their number and viability, as well as the relative content of CD34 ⁺ stem cells among them, are introduced intracerebrally stereotactically under neuronavigation control into the lesion of functionally important structures of the head brain (depending on clinical manifestations), combining with the application of extra-iptracranial microanastomosis in case of def Icyte perfusion of the substance of the brain.

The method is as follows. All procedures associated with obtaining bone marrow aspirate, isolating a mononuclear fraction containing stem cells from it, are performed on the same day as neurosurgical intervention: stereotactic introduction of cells under navigation control into the affected functionally important brain structures (subcortical ganglia, motor zones, visual cortex) , thalamic tubercle). If a deficiency of perfusion of the substance of the brain is detected, this operation is supplemented by the application of extra-intracranial microanastomosis. In the morning, a patient under the conditions of an operating room with anesthetic support by a specialist hematologist performs a trepanobiopsy procedure, during which a bone marrow aspirate in a volume of 200 ml is taken from the iliac wing into a sterile vial with a heparin solution (10,000 units). The vial is transported to the laboratory unit, where it is incubated at 37 ° C for 40 minutes. After gravitational separation of the whole bone marrow, the “upper” fraction is collected in a separate vial, consisting of white suspension cells, which are then centrifuged and precipitated (1200 rpm, 15 min). The autoplasma-containing supernatant is collected in a separate sterile vial, and the cell suspension of the leukemia suspension is resuspended in 40 ml of phosphate-buffered saline containing 10 U / ml heparin (PBS / heparin). Then, the suspension cells are layered on the density gradient of ficoll-verographin (1.078 g / l) and centrifuged for 20 min at 3000 rpm. The bone marrow mononuclear cells (MNC KM) collected from the interphase are washed three times in PBS / heparin and resuspended in 2 ml of physiological saline supplemented with 20% (v / v) autoplasma. For the purpose of laboratory characterization of isolated MNCs, CMs determine their number and viability by trypan blue staining, as well as the relative content of CD34 ⁺ stem cells among them by flow cytometry (FACSCallibur, Becton Dickinson). A properly labeled and sealed bottle with bone marrow cells is transported (on average 2.5-3 hours after trepanobiopsy) to the neurosurgical department, where in the operating room autologous stem cells are injected intracerebrally stereotaxically under neuronavigation control into the lesion of functionally important brain structures.

This stroke treatment method was tested in a clinical setting at the neurosurgical department of the Road Clinical Hospital, Novosibirsk. The proposed method treated 10 patients aged 23 to 67 years with severe persistent neurological deficit caused by ischemic (5 patients) or hemorrhagic stroke (5 patients). Autologous bone marrow stem cell transplantation was performed on average 15 months after the moment of acute cerebrovascular accident. The number of selected bone marrow mononuclear cells varied from 0.8 to 3.0 × 10 ⁹ cells, while the average content of CD34 ⁺ stem cells among them was 100 × 10 ⁶ cells. MNC KM in 2 ml of physiological saline was administered stereotactically under neuronavigation control. Intracerebral transplantation of MNC KM was not accompanied by the development of any complications. All patients showed a significant decrease in spasticity in paretic limbs. In a number of patients, positive neurological dynamics manifested itself in the form of a decrease in the degree of paresis and aphatic disorders. When assessed according to the Barthel scale, an increase in the amount of points by 20% was noted. Thus, the proposed method for the treatment of stroke using autologous transplantation of bone marrow stem cells allows not only to significantly simplify the procedure for obtaining stem cells (by eliminating the steps of isolating the fraction of cells adhering to the plastic, growing them in culture, and directed differentiation in the neuronal direction in the presence of a complex of soluble factors), but also allows to achieve a decrease in neurological deficit in patients in the early or remote period after hay ischemic or hemorrhagic stroke. Moreover, treatment using the proposed method is well tolerated and does not cause the development of any adverse or toxic reactions.

The following are clinical examples that explain this treatment for stroke.

Example 1. Patient S., 30 years old

Diagnosis at admission: Late recovery period after acute cerebrovascular accident (stroke) in hemorrhagic type in the pool of the right middle cerebral artery. Left-side hemiparesis. Episyndrome. Anamnesis: in August 2001, against the background of high blood pressure, there was a hemorrhagic type of stroke. He was hospitalized in the neurological department of Ekibastuz. In the last 12 months. notes the lack of dynamics in the restoration of strength in paretic limbs.

Date of receipt: April 28, 2003.

Date of operation: 05/20/2003 1) trepanobiopsy and obtaining bone marrow aspirate from the iliac wing; 2) autologous transplantation of bone marrow stem cells into the affected subcortical ganglia of the right hemisphere of the brain under neuronavigation control.

Neurological status before surgery: Level of consciousness 15 points on the Glasgow scale. Dysarthria Bradylalia. Left-side lower facial asymmetry (paresis of the central type VII FMN on the left). Tongue deviation to the left (paresis according to the central type of the XII FMN on the left). Rough left-side hemiparesis: the strength in the left hand is 0-1 points, the strength of the extensor-extensors of the forearm is 2 points; strength of the extensor-extensors of the lower leg - 3 points; Strength in the foot - 3 points. Left-sided hemihyposthesia. Pathological symptoms: symptoms of Babinsky, Rossolimo left positive. Abdominal reflexes D> S.

Neurological status 6 months after surgery: Level of consciousness 15 points on the Glasgow scale. Adequate, oriented. Pupils of normal shape and size, D = S, live photoreactions. Light left-side lower facial asymmetry (paresis of the central type VII FMN left). Tongue in the midline. Moderate left-side hemiparesis: strength in the left hand - 2 points; the strength of the extensor-extensor forearm - 3 points, the strength of the extensor-extensor tibia - 4 points, the strength of the extensor of the foot - 3 points, the flexor of the foot - 4 points. Positive dynamics in the form of a decrease in spasticity in the left limbs is noted. Symptoms of Babinsky, Rossolimo on the left are positive. Left-sided hemihyposthesia. Violations of the PTF, meningeal symptoms - not detected.

Among the dynamic changes in the neurological status, one should note a decrease in spasticity in the paretic limbs, as well as an increase in strength in the distal parts of the paretic limbs.

The results of a neuroimaging study:

CT scan of the brain with contrast enhancement from 04/30/03 (at the time of admission): In the subcortical structures of the right parietal lobe, a posthemorrhagic cyst with dimensions of 24 × 20 × 20 mm (9.6 cm ³ ) is visualized.

Brain CT scan with contrast enhancement from 08/21/03 (3 months after autologous bone marrow stem cell transplantation): In the subcortical structures of the right hemisphere of the brain, a posthemorrhagic cyst of a slit shape 20 × 7.7 × 20 mm in size (3.1 cm ³ , i.e., a decrease in the cyst cavity compared to the initial volume by more than 3 times, by 68%).

The results of a neurophysiological study (table 1):

Table 1 Patient S. Before transplantation of MNC KM 10 days after transplantation of MNC KM 2.5 months after transplantation of MNC KM 05/19/2003 05/30/2003 08/07/2003 Latencies left right left right left right Cort 23.80 21.40 22.80 21.40 25.30 21.00 SUBCORT 16.30 16.50 17.10 15.30 14.30 14.70 P14N20 7.52 4.88 5.68 6.16 11.00 6.32 EP-N20 12.60 9.92 11.30 9.60 14.20 9.76 EP-P14 5.12 5.04 5.60 3.44 3.20 3.44 - Transcranial magnetic stimulation (in dynamics): positive dynamics in the form of a decrease in the time of central conducting along motor conductors;

- Somato-sensory evoked potentials (in dynamics): a decrease in the time of central conduction along sensory conductors in the subcortical sections on the right with signs of improved trophism of this section.

Example 2. Patient P., 67 years old

Diagnosis at admission: The early recovery period after stroke in ischemic type in the basin of the left middle cerebral artery. Bilateral occlusion of the ICA, stenosis of the basilar artery. Right hemiparesis. Rough motor, amnestic aphasia. Frequent transient cerebrovascular accidents in the right carotid pool are not medically controlled.

Concomitant diseases: Hypertension III tbsp., Risk IV, frequent atrial premature beats, NK 0.

ONMK date: 12/25/2002

Date of receipt: 01/16/2003

Date of surgery (02/07/2003): 1) trepanobiopsy and receiving bone marrow aspirate from the ilium wing; 2) the imposition of extra-intracranial microanastomosis (EIKMA) on the left; 3) autologous transplantation of bone marrow stem cells into the premotor zone and the region of the posterior part of the lower frontal gyrus, affected by ischemia (fields 6, 44, 45 according to Broadman).

Neurological status before surgery: Level of consciousness 14 points on the Glasgow scale. Amnestic, motor aphasia. Pupils of normal shape and size, D = S, live photoreactions. The movements of the eyeballs in full. Central paresis of the right facial nerve, tongue in the midline. Tendon reflexes from the arms and legs are enlivened on the right according to the pyramidal type D> = S. The tone in the right extremities is elevated according to the pyramidal type. Light right hemiparesis up to 4 points. Symptom of Babinsky, Rossolimo on the right. Violations of sensitivity, meningeal symptoms, disorders of the PTO were not detected. Frequent episodes of deepening neurological deficit throughout the day.

Neurological status 12 months after surgery: Level of consciousness 15 points on the Glasgow scale. Elements of amnestic, motor aphasia. Pupils of normal shape and size, D = S, live photoreactions. The movements of the eyeballs in full. The face is symmetrical, the tongue is in the midline. Tendon reflexes from the arms and legs are enlivened on the right D> = S. The tone in the limbs is normal. Force paresis, pathological signs, impaired sensitivity, meningeal symptoms, disorders of the PTF were not detected.

Among the dynamic changes in the neurological status, a decrease in the severity of aphatic disorders, the disappearance of the pathological pyramidal tone in the right limbs, as well as a complete restoration of strength in the right limbs should be noted.

The results of a neuroimaging study:

Preoperative brain MRI from 01.25.03 (1 month after stroke):

On the left in the frontal lobe (in the middle and lower convolutions), an extensive ischemic focus is visualized (hyperintensive on T2 weighted and hypointensive on T1 weighted) with fuzzy uneven contours of 2.9 × 5.2 × 4.3 cm in size, irregular in shape, heterogeneous in structure. In the white matter of the left frontal and parietal lobes (localized at the level of the bodies of the lateral ventricles) foci were identified (with a tendency to merge), up to 1.6 cm in size, total foci were 1.7 × 4.2 × 2.5 cm, the foci paraventricular (hyperintensive at T2 weighted and hypointensive at T1 weighted).

MRI of the brain from 02/26/2004 (12 months after autologous bone marrow stem cell transplantation): a postischemic cyst of the left frontoparietal region with clear uneven contours, size 3.3 × 3.2 × 4.2 cm, is visualized.

Note: after the autologous transplantation of bone marrow stem cells, there is no positive dynamics in the form of a decrease in the lesion of the brain lesion by 32%.

Transcranial Doppler scanning of cerebral vessels (from 02.21.2004, 12 months after autologous transplantation of bone marrow stem cells): in comparison with transcranial Doppler scanning of cerebral vessels from January 24, 2003, significant positive dynamics in the form of an increase in the linear velocity of blood flow in the middle cerebral artery and anterior cerebral artery of both hemispheres. Compensatory hyperperfusion of blood flow in both posterior cerebral arteries is preserved with a tendency to normalize the velocity parameters of blood flow. A diffuse decrease in vascular resistance in the cerebral arteries is determined. At a depth of 33-38 mm on the left, the EIKMA section with a linear blood flow velocity of up to 34 cm / sec is visualized by color mapping.

The results of a neurophysiological study (table 2):

table 2 Patient P. Before transplantation of MNC KM 14 days after transplantation of MNC KM 13 months after transplantation of MNC KM 02/06/2003 02.21.2003 03/04/2004 Latencies left right left right left right Cort 20.60 20.50 20.60 20.50 20.10 19.50 SUBCORT 15.40 14.60 15.40 15,20 15.00 14.60 P14N20 5.20 5.92 5.28 5.28 5.04 4.88

- Transcranial magnetic stimulation (in dynamics): positive dynamics in the form of a decrease in the time of central conducting along motor conductors;

Thus, verification of the proposed approach under clinical conditions allows us to conclude that this method of treating stroke through the use of autologous transplantation of bone marrow stem cells can achieve a significant reduction in neurological deficit in patients in the early or long-term period after an ischemic or hemorrhagic stroke. Moreover, treatment using the proposed method is well tolerated and does not cause the development of any adverse or toxic reactions.

Literature

1. Chen J., Li Y., Wang L, Zhang Z., Lu D., Lu M., Chopp M. Therapeutic benefit of intravenous administration of bone marrow stromal cells after cerebral ischemia in rats // Stroke. - 2001 .-- Vol.32. - P.1005-1011.

2. Li Y., Chen J., Wang L, Lu M., Chopp M. Treatment of stroke in rat with intracarotid administration of marrow stromal cells // Neurology. - 2001. - Vol. 56. - P.1666-1672.

3. Chen J., Zhang Z., Li Y., Wang L, Xu YX, Gautam SC, Lu M., Zhu Z., Chopp M. Intravenous administration of human bone marrow stromal cells induces angiogenesis in the ischemic boundary zone after stroke in rats // Circulation research. - 2003. - Vol. 92. - P.692-698.

4. Wechsler L.R. Stem cell transplantation for stroke // Cleveland Clinical Journal of Medicine. - 2004 .-- Vol. 71. - Suppl. 1. - S40 - S41.

5. Kondziolka D., Wechsler L., Goldstein S. et al. transplantation of cultured human neuronal cells for patients with stroke // Neurology. - 2000. - Vol. 55. - P.565-569.

Claims (1)

A method of treating a stroke, including intracerebral bone marrow stem cell transplantation during a neurosurgical intervention in the affected area, characterized in that the transplantation is carried out in the early or late recovery period after an ischemic or hemorrhagic stroke, while the whole complex of autologous mononuclear cells is administered as stem cells isolated from bone marrow aspirate, which are used without isolation of individual cell fractions and their cultivation .