Abstract
Successful repair of damage in cerebral palsy (CP) needs effective clinical interventions other than simply symptomatic treatments. To elucidate the feasibility of using olfactory ensheathing cells (OECs) to treat CP in children and adolescents, we conducted a randomized controlled clinical trial (RCT) on 33 patients. The patients were randomly assigned into two groups (treatment group, n = 18; control group, n = 15), and OECs derived from aborted fetal tissue were injected into the bilateral corona radiata in the frontal lobes (a key point for neural network restoration, KPNNR). The Gross Motor Function Measure (GMFM-66) and the Caregiver Questionnaire Scale were used to evaluate the patients' neurological function and overall health status. Among the 14 patients who completed the 6-month study, six received the cell transplantation and the other eight served as controls. In OEC treatment group, GMFM-66 scores were 26.67 ± 25.33 compared with 19.00 ± 20.00 for the control group. Concurrently, the Caregiver Questionnaire Scale score decreased to 77.83 ± 15.99 in the treatment group in comparison to 138.66 ± 64.06 of the control group. This trial, albeit small in sample size, indicates OEC KPNNR transplantation is effective for functional improvement in children and adolescents with CP, yet without obvious side effects. This small-scale study suggests that the procedure may be a plausible alternative method to treat this not yet curable disorder, and we urge further evaluation with a large-scale RCT.
Keywords
Introduction
Cerebral palsy (CP) is a class of brain lesions in children caused by reasons varying from abnormal brain development to perinatal injuries, and manifests in progressive physical dysfunction. Treatment of CP aims at repairing the injured brain and improving functionality. To realize these two aims, a variety of therapeutic strategies have been studied but few of these methods are able to cure this disorder etiologically. Stem cell transplantation has been reported to be effective in animal models, as well as in patients with other degenerative neurological disorders such as stroke and demyelination (26,43, 45); it is thus thought to be promising in treating CP as well given that CP shares many features with these degenerative diseases (2). Hence, to explore the possibility of treating CP with cell transplantation, a well-designed clinical trial would be invaluable to promptly translate bench-side research to bedside treatment (2).
Olfactory ensheathing cells (OECs) are unique glia of the olfactory system that retain exceptional plasticity and can promote olfactory neurogenesis. Because of their relatively easy accessibility in human (8), OECs have become a prime candidate for cell-mediated repair following a variety of CNS lesions. Upon transplantation into these lesions, OECs can stimulate tissue sparing and neuroprotection, enhance outgrowth of both intact and lesioned axons, activate angiogenesis, change the response status of endogenous glia after damage, and remyelinate axons after a range of demyelinating lesions (13–15,19, 20,31,33,34,39,41,42,46). Consequently, cell preparations containing OECs have been widely used to treat patients with spinal cord injury (SCI), amyotrophic lateral sclerosis (ALS), stroke, multiple sclerosis (MS), and other refractory nerve diseases (5–7,17,21,29). Thus, we hypothesize that human OECs hold great promise in treating patients with CP.
We conducted a randomized controlled clinical trial on 33 volunteers to examine whether OECs are effective in treating children and adolescents with CP. This will help to explore an alternative measures to treat the not yet curable disorders such as CP.
Materials and Methods
The clinical trial was conducted at Beijing Rehabilitation Center in conjunction with Beijing Hongtianji Neuroscience Academy in accordance with guidelines issued by the Chinese Ministry of Health (91–006) (28). The families of all eligible patients were properly informed about the nature of the study, and parents or legal guardians provided written informed consent for patients to participate in this trial.
Patient Enrollment
Patients ranging from 1 to 12 years old diagnosed with definite CP were eligible for inclusion in the study. Exclusion criteria included: 1) taking any experimental medicine and/or participating in any experimental treatments within 1.5 years; 2) any medical condition that may interfere with the Gross Motor Function Measure (GMFM-66) and the Caregiver Questionnaire assessments; 3) known clinically significant neurological, musculoskeletal, cardiovascular, respiratory, hepatic, or renal disease and/or malignancy; 4) hemophilia or other bleeding abnormality; 5) diabetes mellitus requiring insulin therapy; 6) known immunodeficiencies, including acquired immune deficiency syndrome or use of immunosuppressive or cancer chemotherapeutic drugs; 7) any condition or situation likely to cause patients to be unable or unwilling to participate in study procedures or participate in all scheduled study assessments, including follow-up through the sixth month of the study; 8) any preexisting condition likely to result in the patient's death within the next 12 months; 9) any infections at the site of surgery; any condition or situation likely to cause the patient to be unable to undergo rehabilitation procedures; 10) patients who lacked family support were also excluded because the study design was predicated on the assistance of caregivers to perform assessments.
Between October of 2006 and May of 2008, 33 patients who met the following inclusion criteria were enrolled in this clinical trial: age of 1–12 years old; either male or female; diagnosis of CP; Informed Consent Form signed by the parents or patient's legal guardians; the patient, parents, or patient's legal guardians are able to communicate effectively to obtain informed consent and to ensure neurological examination. They were randomly assigned to two groups (treatment group, n = 18; controls, n = 15), according to the randomized allocation table, which was designed for the total of 40 subjects to be recruited in the protocol of this clinical trial.
Cell Preparation
OECs were isolated and cultured as described previously (18). Briefly, OECs were isolated from aborted human fetal olfactory bulb with properly consent of the donors. The OECs were cultured and propagated for 2–3 weeks and characterized by immunostaining with antibodies against p75 (a neurotrophin receptor that is specific for OECs; Sigma) (18) (Fig. 1). OECs from one to two fetuses, which represent 2 million cells, were transplanted for each patient. HLA-DR-matching test was carried out before transplantation to ensure the histocompability between the donors and the recipients.
Immunostaining by human-specific p75 (soma and branching) and Hoechst (ovals) antibody identifies olfactory ensheathing cells in culture.
Implantation of Fetal OECs
White matter injury or periventricular leukomalacia (PVL) are shown with T2 weighted and fluid attenuated inversion recovery (FLAIR) MR images. Two million cells in 100 μl of medium were injected into the bilateral corona radiata of the frontal lobes (key point for neural network restoration, KPNNR) using stereotactic techniques under local anesthesia (Fig. 2). No participants received immunosuppressant therapy before or after surgery.
MRI films show the targets of cell transplantation: two injection sites of OECs in the corona radiate of both-sided frontal lobes [i.e., the key point for neural network restoration (KPNNR)].
Measurement of Outcome and Follow-Up
The neurological function of patients was evaluated by three neurologists in a double-blind fashion. The Gross Motor Function Measure (GMFM-66) and Caregiver Questionnaire Scale, together with a video record of each patient were taken as scheduled. Neurological examination and magnetic resonance imaging (MRI) scan were recorded within 1 month before the scheduled surgery. Eligible participants were randomly assigned to either treatment or control groups. Patients of both groups got nonaltered physiotherapy and a home training program for the next 6 months. All participants were followed up for the following 6 months after the initial evaluation at baseline or surgery.
Statistical Analysis
The data were expressed as means ± SD. The data were analyzed with SPSS for Windows (version 10.0; SPSS, Inc., Chicago, IL), for either two-tailed independent sample t-test, paired samples t-test, or chi-square test. A significance level of 0.05 was used.
Results
OEC Transplantation Is Safe for Recipients
All participants survived the transplantation procedure, although some were lost to follow-up because of difficulties in long-distance travel. Fourteen patients (six in the OEC-treated group and eight in the controls) completed this study over a period of 6 months. No patients in the OEC-treated group experienced side effects or complications following the operation. These results suggest that OEC transplantation is a safe procedure for treating CP.
OEC Transplantation Is Effective for CP
In the treatment group, there were three boys and three girls, ranging from 2 to 12 years old (5.3 ± 3.8 years) at enrollment. The control group comprised five boys and three girls of similar age (2.5–10 years old, 6.6 ± 2.5 years) at enrollment. Patients of the two groups were comparable at baseline with respect to age [χ2(1) = 0.219, p = 0.640], sex (t = 0.767, p = 0.458), and parameters such as GMFM-66 and Caregiver Questionnaire Scale. The GMFM-66 score at entry was 19.00 ± 20.00 for the treatment group and 36.75 ± 27.14 for the controls (t = 1.346, p = 0.203); the Caregiver Questionnaire Scale score at entry was 138.66 ± 64.06 for the treatment groups and 143.75 ± 47.20 for the controls (t = 0.172, p = 0.867).
At the end of the 6-month follow-up period, the GMFM-66 score of the OEC treatment group was 26.67 ± 25.33, which is higher than 19.00 ± 20.00 of the control (t = −2.823, p = 0.037). The Caregiver Questionnaire Scale score was 77.83 ± 15.99, much lower than the 138.66 ± 64.06 of the control group (t = 2.100, p = 0.090). For the controls, the GMFM-66 score decreased from 36.75 ± 27.14 to 33.75 ± 25.44 (t = 1.101, p = 0.307) with no significant difference, and the Caregiver Questionnaire Scale score decreased from 143.75 ± 47.20 to 119.25 ± 40.33 (t = 2.229, p = 0.061).
Over a period of months, we also observed progressive changes of the two parameters in both groups, yet the rate of GMFM-66 was 7.67 ± 6.65 in the OEC treatment group, higher than the −3.00 ± 7.71 for the controls (t = −2.710, p = 0.019). Similarly, differential rates of Caregiver Questionnaire Scale score were also obtained in the treatment and control groups, which were 60.83 ± 70.97 and 24.50 ± 31.08, respectively (t = −1.173, p = 0.282) (Tables 1 and 2). In summary, we concluded OECs help to improve the neurological function of CP patients over time.
Clinical Data of Subjects in the Treated Group
Clinical Data of Subjects in the Control Group
Discussion
By conducting this prospective random controlled clinical trial, we provided the evidence of the feasibility and efficacy of OECs in CP patients. Our result clearly showed that transplanting OECs into CP patients improved the neurological function of the recipients, and did not cause significant side effects. The failure to differentiate the improvement by Caregiver Questionnaire Scale score was most likely due to the small sample size, which will be overcome by incorporating more patients in the future trials.
Pathologic Finding in CP
In this study, we have frequently observed tissue loss (i.e., necrosis and atrophy), periventricular leukomalacia, inadequate and/or delayed myelination, glial scars, shrinked white matter, and encephalomalacia in the CP child. This was in accordance to the previous reports of severe hypoxic-ischemic injuries in multiple sites of the brains, such as the medial cerebellar hemispheres, medial temporal lobes, thalami, and corona radiata, which were always found on the MR imaging (12,40). Our findings are also in agreement with studies by Korzeniewski et al., which indicated that most (83%) children with CP have abnormal neuroradiological findings, with white matter damage the most common abnormality. Combined gray and white matter abnormalities are more common among children with hemiplegia; isolated white matter abnormalities, however, are always found with bilateral spasticity or athetosis, and with ataxia. Isolated gray matter damage, as reported, is the least common finding (11).
Possible Mechanism of OECs Effect
OECs have the capability to continually support the regeneration of olfactory receptor neurons throughout life. These olfactory receptor neurons are unique for their potential to extend primary axons into the central nervous system (CNS) during adulthood, which represents one of the rare instances of the continual neurogenesis in the peripheral neurons system (PNS) (1). Human OECs that were derived from fetal tissue have been shown to have a remarkable ability to protect neurons in various types of disorders, such as spinal cord injury (9,24,44), stoke (35,36), peripheral nerve injury (30), optic nerve injury (16), amyotrophic lateral sclerosis (22), cognitive dysfunction (37), demyelinating diseases (38), Parkinson's disease (25), hearing loss (4), and retinopathy (23). These OECs, when transplanted into adult mammalian CNS, were able to produce Schwann cell-like myelin sheaths around demyelinated axons (10). In fact, transplantation of this kind of ensheathing glia can also promote axonal regeneration within the adult CNS (32). It was thought that OECs did these in PNS and CNS by similar mechanisms such as enhancing axonal regeneration (15), releasing neurotrophic factors to support injured neurons (27), and through accelerating angiogenesis and remyelination of axons (3). This study indicates that OECs improved the neurological functions of CP patients through similar restorative effects as those reported previously.
A Hypothesis of Key Point for Neural Network Restoration (KPNNR)
Diseases of the central nervous system, such as CP, ALS, ataxia, MS, dementia, and epilepsy, although they vary in etiology and pathologic findings, ultimately affect similar elements in the brain such as neurons and nerve fibers of the cerebral cortex, spinal cord, cerebellum, pons, brain stem nuclei, red nucleus, substantia nigra, and basal ganglia. Thus, there might be a key point among these elements where cell graft should be delivered for cell-based therapy. KPNNR was first proposed in 2003 by Huang et al. after a set of successful cases in clinical practice (7). According to this theory, the key target where the cells should be transplanted is located at anterior 1/4–1/3 of ateral ventricle and 23–27 mm away from the midline. This site is where the frontal corona radiata pyramidal tract pass through, and represents a point at which numerous projection fibers, association fibers, and commissural fibers convergent. After transplanted into this important “point” in the brain, the cells will initiate an extensive bidirectional remodeling in the entire neural network, including cerebrum, cerebellum, and spinal cord. Thus, the key point of cell transplantation should be of great importance in approaching the functional neurorestoration. In combination with other treatments such as drugs, growth factors, and genetic therapies, our method should help significantly in approaching an effective cure of CP.
Footnotes
Acknowledgments
We thank Richard Douglas Mcguiness, Soren Laursen, Baoyang Hu, and Priscilla Song for preparing this manuscript.