Stem Cell Treatment For Severe Head Injury (traumatic Brain Injury)

Biomedicine & Pharmacotherapy 59 (2005) 415–420 http://france.elsevier.com/direct/BIOPHA/

Original article

Cell transplantation therapy in reanimating severely head-injured patients Victor I. Seledtsov a,*, Samuil S. Rabinovich b, Oleg V. Parlyuk c, Marina Yu. Kafanova c, Sergey V. Astrakov c, Galina V. Seledtsova a, Denis M. Samarin a, Olga V. Poveschenko a a

Immunohematologic Department, Institute of Clinical Immunology, Russian Academy of Medical Sciences, 14 Yadrintsevskaya Street, 630099 Novosibirsk, Russia b Novosibirsk State Medical Academy, Novosibirsk, Russia c 34 Municipal Clinical Hospital, Novosibirsk, Russia Received 14 January 2005; accepted 31 January 2005 Available online 07 July 2005

Abstract The results of controlled, retrospective clinical investigation of applying cell transplantation (CT) therapy in 38 severely head-injured patients are presented. The patients initially were in state of coma (Glasgow coma scale score 3–7), owing to their traumatic brain injuries. Cells prepared from fetal nervous and hematopoietic tissues were grafted subarachnoidally via lumbar puncture. The control group consisted of 38 patients and was clinically comparable with the trial one. From the results obtained it appears that CT treatment promoted both wakening consciousness of the patients and their following neurological rehabilitation. A death-rate in the trial and control group was 5% (two cases) and 45% (17 cases), respectively. According to a Glasgow scale, favorable (good + satisfactory) outcomes of a disease were noted in 33 (87%) cell-grafted and only in 15 (39%) control patients. Statistical analysis revealed that CT treatment generally improved the outcomes by 2.5fold. No serious complications of CT therapy were noted. The results point out a possible rationality of applying CT therapy in severely head-injured patients as early as within acute period of a disease. © 2005 Elsevier SAS. All rights reserved. Keywords: Cell transplantation; Brain injury; Coma

1. Introduction A severe head-injury remains is one of main reason for mortality and disability among able-bodied citizens. Outcomes of treating severely head-injured patients are largely defined within in an acute period of a disease. In this period medical interventions are aimed at preventing the injuretriggered, second pathological processes that result in additional damages of a brain tissue and are frequently associated with life-threatening complications. Clinical effects of neuroprotective drugs in acute brain-injured patients are often

Abbreviations: BC, brain contusion; CNS, central nervous system; CT, cell transplantation; DAI, diffuse-axonal injury; EEG, electroencephalography; EH, epidural hematoma; GCS, Glasgow coma scale; IH, intraventricular hematoma; MRI, magnetic resonance imaging; SH, subdural hematoma; TUDG, transcranial ultrasonic dopplerography. * Corresponding author. Tel./fax: +7 3832 28 2673; fax: +7 3832 22 7028. E-mail address: [email protected] (V.I. Seledtsov). 0753-3322/$ - see front matter © 2005 Elsevier SAS. All rights reserved. doi:10.1016/j.biopha.2005.01.012

unclear and doubtful (reviewed in [10]), and there is an apparent necessity to search new approaches to recovery of lifesaved, brain functions in such patients. Cell-based technologies allowing to repair injured organs at a cellular level open up fundamentally new opportunities in treating many problem diseases, including neurological ones. The central nervous system (CNS) is an “immuneprivileged” organ where there are substantial barriers to developing alloantigen-induced, immune processes. In fact, the grafted neural cells have been convincingly documented to be able to survive in the major histocompatibility complex (MHC)-incompatible CNS for relatively long period of time. There is also ample evidence from various experimental studies indicating abilities of the transplanted cells to proliferate and elaborate cell growth factors in brain lesions and to markedly intensify, thereby, brain tissue reparation processes (reviewed in [2,4]). In this paper we present the results of applying a subarachnoidal fetal cell transplantation (CT) in 38 acute, severely

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head-injured patients with a high risk of a poor outcome of a disease. 2. Materials and methods The study was performed in the exact accordance with the protocol approved by the Scientific Council and Ethics Committee at the Institute of Clinical Immunology. Informed consent was obtained from the closest relations of each subject who has been enrolled in the study. The fetal brain neural and hemopoietic liver tissues were isolated from human fetuses (gestational age 16–22 weeks) after spontaneous or therapeutic abortion, and then prepared in the form of cell suspension, as described earlier [16]. The cells were further cryopreserved in the standard way in 90% fetal bovine serum containing 10% dimethyl sulfoxide, and stored in liquid nitrogen until use. On the day of transplantation, the cell suspensions were thawed at 37 °C, washed extensively, and assayed for cell viability by a erythrosine exclusion method in the routine way. The overall number of viable cells in the suspension intended for a single administration was 2.0 × 108; the neural to liver tissue cell ratio in such suspension was of 10:1. The cells were grafted subarachnoidally via lumbar puncture. Thirty-eight patients (10 females and 28 males) aged from 18 to 63 years (an average age 38) have been enrolled in the study. These patients were admitted to the clinic in a state of coma, owing to severe traumatic brain injury. We did not enter onto the study the patients who had extracranial injuries which, by themselves, were life-threatening. Glasgow coma scale (GCS) scores of trial patients were in the range of 3–7. A diffuse-axonal injury (DAI) was diagnosed in 23 (60%) patients that in 19 (50%) cases was compatible with a hematoma-associated brain compression. In the remaining 15 (40%) patients there was a severe brain contusion that also associated with a brain compression. In all patients a brain compression was remedied in an emergency order. The further intensive therapy allowed the patients to stabilize their cardiovascular and respiratory activities. However, in spite of all therapeutic interventions, the patients did not recover their consciousness. In these cases a magnetic resonance imaging (MRI) typically revealed diffuse-atrophic alterations of both white and gray brain matter; an electroencephalography (EEG) demonstrated the strong decrease in functional brain activity and the disappearance of a-rhythm; a transcranial ultrasonic dopplerography (TUDG) exhibited the significant reduction in linear brain blood flow velocity. In general, the state of the patients was characterized by a high risk of developing a long-term vegetative status and lethal outcome. CT treatment was undertaken when consciousness of a patient did not exhibit signs of its recovering as long as at 5–8 weeks post-injury. Twenty-five patients were cellgrafted once. Other 12, and one patients were cell-grafted twice, and thrice, respectively, at a 10–14 day interval. The control group included 38 patients aged 19–60 years (an average age 38) and was formed retrospectively on a pair

basis. Each control patient was randomly selected to be a clinically comparable counterpart of a trial patient (Table 1). The median GCS score in the control and trial group was of 4.6 and 4.1, respectively. Both the control and trial patients received a similar standard therapy in equivalent conditions during the same time. Clinical outcomes for both trial and control patients were assessed in terms of the Glasgow outcome classification at 18–24 months post-injury. For statistical analysis, it was accepted that a lethal, unsatisfactory, satisfactory, and good outcome was coincided with 0–3 points, respectively. A paired Student’s test was used to determine the significance of differences between trial and control values. 3. Results In 33 of 38 trial patients the signs of awakening consciousness in the form of opening eyes and performing the simplest tasks occurred as early as at 3–7 days post-grafting. During following 5 days those patients became contacting their relations and a medical personal. A restoration of their main psychical functions was observed at 15–20 days after CT treatment. By that time an a-rhythm appeared and a brain blood flow attained a lower limit of the norm. The other three cell-grafted patients also exhibited awakening consciousness after CT treatment. However, they further retained significant defects in their psychoemotional sphere and were in need of an extraneous assistance. Those patients were cell-grafted once again. The appreciable benefits from CT treatment were noted in those cases. Nevertheless, these subjects remained neurological defects that significantly limited their functional abilities. The remaining two cell-grafted patients exhibited only some signs of awakening consciousness after CT treatment. In spite of all medical interventions undertaken, they both died later from extracranial complications. With CT treatment positive changes in stem brain symptoms were noted in the patients, indicating restoration of their vitally important, brain functions (Table 2). On the whole, as shown in Table 3, CT treatment enabled to considerably decrease a death-rate among severely headinjured persons and to increase a proportion of the patients with favorable (good + satisfactory) clinical outcomes. As shown in Fig. 1, the outcome value (M ± m) for CT-treated patients exceeded the analogous value for control patients by 2.5-fold (P < 0.001). No significant changes on MRI scans of the patients was typically observed within acute period of disease. However, 1–1.5 years later MRI signs of brain atrophy almost completely disappeared in all patients with favorable outcomes of a disease (Fig. 2). By present, the follow-up time for 25 cell-grafted patients is of 4–6 years. Of these 20 persons were ultimately rehabilitated to an extent to be able to continue their working activity in full measure. No CT-related complications was noted over the whole follow-up period.

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Table 1 Patients’ characteristics

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38

Patient, age, sex P., 49, ? R., 19, 4 M., 24, 4 V., 18, ? Sh., 34, 4 B., 29, 4 B., 24, ? M, 56, ? D, 18, 4 Ch, 32,4 Ch, 38, ? M., 48, ? P., 63, ? Ch., 52, ? K., 19, 4 S., 36, ? M., 48, ? L., 44, ? R., 35, ? R., 35, ? A., 32, ? K.,45, ? N., 46, ? A., 54, ? S., 45, 4 D., 43, ? M., 35, ? K., 29, 4 S., 34, 4 L., 34, ? K., 45 ? Ch., 47, ? A., 47, ? R., 43, ? V., 23, ? N., 34, ? L., 56, ? G., 17, ?

Trial Brain injury DAI, SD d DAI DAI, SD d BC, IH BC, SD d DAI, SD d BC, EH d BC, SD d DAI, SD s DAI DAI, SD d,s DAI BC, SD d BC, SD s DAI DAI, IH DAI, SD s BC, SD s BC DAI, IH DAI, SD d BC, IH BC, SD s DAI, SD s DAI, IH BC, EH d DAI, IH DAI, SD s BC, SD s DAI, SD s BC, SD d DAI, IH DAI, SD s DAI, IH DAI, SD s BC, SD s DAI, IH BC

GCS score 4 5 5 7 7 4 5 6 4 3 3 5 5 4 3 4 5 4 3 3 4 5 4 3 3 4 4 3 7 5 7 3 4 4 6 7 5 7

Patient, age, sex U., 43, 4 Sch., 33, 4 M., 32, ? R., 23., ? G., 41, ? B., 28, ? S., 23, ? V., 56, ? U., 19, ? P., 56, ? R., 31, ? Ch., 39, ? P., 53, ? Ch., 49, ? R., 19, 4 D., 45, ? P., 60, 4 P.,.37, ? T., 28, ? T., 38, ? G., 29, ? K.,43, ? G., 41, ? P., 53, 4 E., 45, 4 Sch., 53, ? M., 32, 4 E., 34, ? V., 23, 4 R., 27, ? N., 49, 4 G., 41, ? Ch., 36, ? P., 23, 4 G., 47, ? S., 34, ? F., 39, ? O., 33, ?

Control Brain injury DAI, SD d DAI DAI, SD d BC, IH BC, SH d DAI, SH d BC BC DAI, SH s DAI, IH DAI, IH DAI, IH BC, SD s BC, SD s DAI, IH DAI, IH DAI, BC BC DAI, SD d BC, SD d BC, SD s DAI, SD d DAI, SD s DAI, IH DAI, SD d DAI, SD d DAI, SD s BC, SD s DAI, SD s DAI, SD d DAI, SD d BC, SD s BC, IH BC, SD s BC, SD d BC, SD d BC, SD s

GCS score 4 4 5 5 5 4 4 6 3 3 3 5 4 4 3 4 3 4 4 3 4 4 3 3 4 4 3 3 6 5 3 3 4 5 3 6 5 7

The used abbreviations are: BC - brain contusion; DAI - diffuse-axonal injury; EH - epidural hematoma; IH - intraventricular hematoma; SH - subdural hematoma.

Two cases of applying CT are described in detail below. Table 2 Stem brain symptoms in the patients (n = 38) before and at 12–15 days after CT treatment Symptom

Quantity of patients (%) Before After treatment treatment Respiratory disorder 24 (63) 0 Lack of swallowing reflex 38 (100) 0 Extrapyramidal tetrasyndrom 29 (76) 3 (8) Impairment or lack of corneal reflexes 38 (100) 0 Impairment or lack of iris contraction reflex 24 (63) 3 (8) Paresis of look upward 29 (76) 4 (10) Oculocephalic reflex: Lack 6 (16) 0 Impairment 21 (55) 3 (8)

3.1. Case 1 An 18-year-old female patient D was injured in a vehicular accident. On admission her pulse rate was 120–128 bpm, arterial blood pressure 100/60; there was a psychomotor excitation, hyperhidrosis, and hyperthermia (up to 40 °C); a Table 3 Outcomes of treating a severe brain injury Outcome Lethal Unsatisfactory Satisfactory Good

Trial (n = 38) 2 (5%) 3 (8%) 15 (40%) 18 (47%)

Control (n = 38) 17 (45%) 6 (16%) 15 (39%) 0

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3.2. Case 2

Fig. 1. The outcome values (M ± m) for trial and control patients.

depressed fracture of temporal was seen on the right. Because of inefficiency of self-dependent respiration, the patient was transferred on artificial pulmonary ventilation. Her GCS score was 4. On MRI a subdural hematoma was revealed on the left; cisterns and ventricles of the brain were not visualized. The hematoma was removed in a surgery way. Intensive therapy enable the patient to normalize her vital functions including respiration. However, in spite of all medical interventions undertaken, patient’s consciousness was not recovered. For this reason, the patient was cell-grafted on 37 and 48 days postinjury. Signs of awakening patients’s consciousness appeared as early as at 4 days after the first CT. On 7 days after the second CT the consciousness was recovered to the level of light obnubilation. Three months later a completed recovery of her psychical functions was noted under a control examination. As early as at 6 months after CT treatment, MRI signs of her brain atrophy almost completely disappeared (Fig. 1A, B). The Glasgow outcome of her disease was good. At 1.5 years post-injury she became a student of the university faculty. By the time of preparing this manuscript she was an excellent student in her third year.

A 24-year-old male patient B was admitted to the Emergency City Hospital after a vehicular accident. On admission his pulse rate was 110 bpm, arterial blood pressure 150/90; respiration was superficial, arrhythmical, at 28 per min; there was a psychomotor excitation with periodic hormetonic convulsions. His GCS score was 5. The patient was transferred on artificial pulmonary ventilation. MRI revealed an intracranial hematoma in the right temporoparietal area. This hematoma (120 ml) was removed in a surgery way. Intensive therapy enable the patient to restore adequate self-dependent respiration on 5 days after trauma. A repeated MRI revealed contusion focuses of III type in frontotemporal-basilar brain areas. In spite of conducting intensive rehabilitation therapy, the patient did not recovery his consciousness over 28 days. The patient was cell-grafted twice on 28 and 40 days postinjury. Recovering patient’s consciousness to the level of light obnubilation occurred as early as on 6 days after the last CT. Recovery of his directional sense was noted 5 days later, whereas recovery of his time sense took significantly more time. The patient was discharged on 52 days post-injury. The Glasgow outcome of his disease was good. Three years later he became a student of the university faculty, successfully managing his educational task.

4. Discussion The results presented herein suggest that cells from nervous and hemopoietic fetal tissues, when subarachnoidally grafted, are able to promote recovering useful consciousness of a severely head-injured patient. Patient’s consciousness

Fig. 2. The MRI scan of the patient D, 18 years old, before (A) and at 6 months after CT treatment (B). For description see text.

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awakening, by itself, may be an important trigger signal for activation of multiple mechanisms which are capable of both reducing an incidence of potentially life-threatening complications and ameliorating neurological functional defects. Since apparent sighs of recovering of patient’s consciousness typically occurred as early as within 7 days after CT treatment, the effects of a CT therapy on brain functionality in this period are most likely due to a release by grafted cells of mediators stimulating coordinative work of various brain structures. This suggestion is consistent with the published data indicating an ability of neural progenitor cells to elaborate essential neurotrophic factors and promote, thereby, both survival and functionality of degenerating neurons after traumatic brain injury [6]. As shown in this paper, CT treatment not only reduce a death-rate of severely head-injured patients but also substantially increased proportion among them of persons with favorable outcomes of a disease. To our opinion, the latter might be explained by a long-term influence of grafted cells upon reparative processes occurring in a nervous tissue in response to injury. As a matter of fact the brain is a plastic system able to integrate transplanted, fetal-derived, allogeneic stem/ progenitor cells. On one hand, donor cells may be longacting producers of neurotrophic mediators, on the other hand they may be directly implicated in newly forming nervous communications (reviewed in [2,4]). A subarachnoidal pathway of cell transplantation into CNS is safe and well tolerated. As experimentally shown [19], the cells of immature nervous tissue, when grafted within subarachnoidal cavity, are capable of migrating into brain lesions and intensifying there reparative processes. An effective CNS repair requires the presence in injured sites of not only neural cells potentially able to provide axonal growth, but also the other cells capable of creating the microenvironment favorable to both growth and myelination of nerve fibers. In fact, schwann cells grafted in a brain lesion have been found to be able to stimulate axonal growth [1]. In a brain lesion donor oligodendrocytes can synthesize a myelin [9], whereas donor astrocytes are capable of inhibiting the development of a glial scar tissue [8,13,18] that can become a insuperable obstacle to axonal growth. In our own investigation we transplanted into the patients not only the cells isolated from immature nervous tissue, but also the fetal liver cells. The human liver of gestational age of 16–22 weeks is a hematopoietic organ with relatively high contents of immature multipotent cells [5]. Evidence is accumulating that fetal hematopoietic tissues contain the stem cells capable of ameliorating functional neurological defects [15]. These cells are able to be differentiated into neurons and astrocytes [3,7], and may contribute to neovascularization of ischemized tissues [12]. Moreover, they are able to inhibit scar connective tissue development [11]. In general, to our opinion, the cell transplantat composed of various types of stem and progenitor cells may be much more effective in repairing an injured tissue in comparison with the transplantat consisting only of one type of stem or progenitor cells.

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Noticeable benefits from CT-based technologies in treating have been previously noted in patients with ultimate (chronic) consequences of traumatic brain injury [14,17], when not only prime, but also second, injury-induced, pathological processes may be already developed. Induction of donor-specific immune reactions was found in part of these patients. At the same time no laboratory and clinical signs of developing tissue-destructive, autoimmune processes were observed [17]. The date presented in this paper suggest clinical reasonability of using CT therapy for a severely head-injury as early as within acute period of a disease, when a patients is unconscious. Such timely treatment is likely to be able to prevent/reduce the development of the second pathological processes that are disabling and potentially life-threatening. Importantly, no serious complications which might limit application of CT-based technologies in head-injured patients were noted. On the whole the results presented in this paper are undoubtedly promising. Although much greater clinical experience is needed to determine a place and clinical relevance of CT-based therapy in overall complex treatment of the patients with brain. It is reasonable to anticipate that developing CT-based approaches may provide much progress in the management of multiple neurological diseases including those which are now considered as uncurable. Novel techniques of preparation and propagation of multi- and unipotent cells, which are being now actively developed, enable to solve not only technical, but also ethical problems confronting progress in cell transplantology (reviewed in [3]) and, thereby, may promote widespread adoption of CT-based advances in clinical practice.

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