Traumatic brain injury (TBI) is a major cause of death (50,000/yr) and disability. The Centers for Disease Control and Prevention (CDC) has estimated that approximately 1.4-1.5 million Americans survive TBI annually. The economic burden of TBI in the United States has been estimated at approximately $56.3 billion in 1995. In children TBI has lifelong cognitive, physical, psychosocial/ behavioral/ emotional impairments since it interferes with the developing brain. In fact, in children deficits may not be fully revealed until later in life. TBI is among the most frequent pediatric neurological conditions (400,000 emergency department visits/yr; Langlois, 2001), occurring more frequently than cerebral palsy (10,000/yr), global developmental delay (80,000/yr) and epilepsy (30,000/yr). TBI continues to pose a serious health concern and adequate treatment for these injuries is seriously lacking. For example, the majority of TBI patients are discharged without subsequent health-care assistance. Among children aged 0-4 years 91% are unattended clinically post discharge (compare to 66% for all injuries). This is particularly discordant with data showing that at least 15% of TBI patients continue to experience negative consequences 1yr after injury (Guerrero et al, 2000). Optimistically, the neonatal brain may be more amenable than the adult brain to the therapeutic potential of stem cells. Transplantation studies using human embryonic stem cells and other stem cells have demonstrated their ability to facilitate functional recovery after brain and spinal cord injury. The proposed studies will determine the therapeutic capacity of neuron-enriched human embryonic stem cells (hESCs) to attenuate histological damage, normalize brain metabolite profiles, generate functional neural circuits and recover sensorimotor and memory functions following transplantation into postnatal rats subjected to lateral fluid percussion injury (FPI). The lateral FPI model most closely mimics postlesional events associated with TBI in humans (Dietrich et al, 1996). In Aim 1 we will evaluate whether hESCs (previously differentiated into neurons and labeled ferromagnetically) transplanted into the traumatically injured pediatric brain migrate to the site of injury and compensate for lesion area and brain metabolite changes using brain imaging methods. In Aim 2 accelerating rotarod and Morris water maze tests will be used to investigate whether grafted hESCs can ameliorate sensorimotor and cognitive deficits. In Aim 3 we will use immunohistochemical methods to label neural and glial phenotypes and synaptic contacts to determine if grafted hESCs form functional neuronal networks in injured brain. Our findings should eventually help promote the development of novel strategies to alleviate the effects of TBI. The proof of concept obtained in these studies will provide the necessary preliminary data and expertise necessary to be competitive for NIH funding.
Statement of Benefit to California:
Traumatic brain injury (TBI) is a major cause of death (50,000/yr) and disability. The Centers for Disease Control and Prevention (CDC) has estimated that approximately 1.4-1.5 million Americans survive TBI annually. In contrast, the incidence of breast cancer and HIV/AIDS is 8 and 34 times lower (National Center for Injury Prevention and Control, CDC, http://www.cdc.gov/ncipc/factsheets/tbi.htm). The economic burden of TBI in the United States has been estimated at approximately $56.3 billion in 1995. In addition, TBI imposes enormous losses to individuals, their families, and society that cannot be completely enumerated. TBI is among the most frequent pediatric neurological conditions (400,000 emergency department visits; Langlois, 2001), occurring more frequently than cerebral palsy (10,000/year), global developmental delay (80,000/yr) and epilepsy (30,000/yr). TBI also accounts for significant neurological and neuropsychological morbidity in children. Data from 12 states, including California, indicate that the age-adjusted rate for TBI-related hospitalizations in 2002 was 79.0 per 100,000 population. While this estimate is ~20% lower than annual estimates for 1994-1995 from the National Hospital Discharge Survey (NHDS), TBI continues to leave an estimated 80,000-90,000 persons with long-term disability (Langlois et al, 2004). This apparent decline should be interpreted cautiously as estimates exclude non-residents and brain injuries not treated at hospitals. Most TBI cases are mild and deficits are subtle. In children deficits may not be fully revealed until later in life. TBI, which produces cognitive, physical and psychosocial/ behavioral/ emotional impairments, that often continue to develop for up to months and years after impact, continues to pose a serious health concern and adequate treatment for these injuries is seriously lacking. For example, the majority of TBI patients are discharged without subsequent health-care assistance. Among children aged 0-4 years 91% are unattended clinically post discharge (compare to 66% for all injuries). This is particularly discordant with data showing that at least 15% of TBI patients continue to experience negative consequences 1yr after injury (Guerrero et al, 2000). Optimistically, neonatal brain injury may be more amenable than the adult brain to the therapeutic potential of stem cells (Santner-Nanan et al, 2005). Transplantation studies using non-embryonic stem cells from human fetus, mouse embryonic stem cells have demonstrated their ability to facilitate functional recovery after brain and spinal cord injury. hESCs offer greater ability of pluropotency and can prevent any cross-species consequences. To date a very limited number of studies have explored the ability of hESCs to reverse or protect against detrimental and permanent effects of TBI, especially as measured noninvasively using brain imaging.
SYNOPSIS: This proposal is designed to examine the potential therapeutic role of human embryonic stem cells (hESC’s) in traumatic brain injury of the neonate. The applicant makes the point that the neonatal brain may be more amenable to stem cell therapy than the adult. The applicant plans three specific aims. In Specific Aim 1, they will use neural stem progeny of hESC’s labeled with iron oxide nanoparticles. These cells will be transplanted into the ventricles or parenchyma of PN 10 rats that had been subject to mild cortical injury. MRI and MRS will then be used to study the migration of cells and metabolic changes associated with them. In Specific Aim 2, they will use a battery of behavioral testS to explore the effects of therapy on sensorimotor and cognitive deficits in treated and control animals. In Specific Aim 3, they will explore the ability of the transplanted cells to form neural networks in the transplanted brain, as determined morphologically. SIGNIFICANCE AND INNOVATION: One reviewer felt that the use of hESC derivatives in treating human traumatic brain injury would clearly be novel and clinically important given the prevalence of the problem and its cost to society. This proposal would use contemporary cell tracking techniques to follow cells after transplantation. The strategies to be used are relatively straightforward and utilize new approaches in cell tracking. The PI has proposed to follow the migration and integration of iron oxides labeled hESC derived neural stem cells by MRI in cortical contusion injury (CCI) rat model. PI will also assess the functional recovery of the injured rats at different interval after CCI. Changes in lesion volume, status of migrated cells and metabolic activity in the lesion following cell implantation will be assessed by T2WI, DWI, MRS and or SWI sequences at days 1, 3, 7, 14, 30, 60 and 90. Functional recovery will be assessed by well established cognitive and motor function tests. The proposal seems to be a cut and paste from other submitted protocol. PI is calling herself as co-PI in the section of “Feasibility”. Some of the figures do not match with the corresponding descriptions. Another reviewer felts that the proposal is moderately innovative and significant. As described by the investigator TBI is one of the major causes of death and disability. Minor TBI during childhood is especially overlooked and can have a devastating consequence at later part of life. Due to multi-potential capability, hESC derived neural stem cell can help repair the injured tissues either by involving directly or through trophic factors. SVZ derived neural stem cells in different brain injury models have been used, however, use of hESC derived neural stem cells is limited. This study will broaden the data needed to go for future human trials. STRENGTHS: The proposal brings together two scientists, Drs. Curras-Collazo and Obenaus who have complementary skills. The PI’s background is in neuroendrocrinology but she has early training in hESC research. Dr. Obenaus has quite extensive experience in brain injury and MRI. They collectively have shown evidence that they are capable of doing the imaging work and some of the early stem cell culturing. They have an experienced stem cell collaborator – Dr. N. Sato. Another reviewer felt that a strength of the proposal is the inclusion of experts of stem cell biology and MRI. The PI herself is an expert in neuroscience. Apart from few flaws in the study protocols the study is well designed and can be accomplished after correcting and re-writing the protocols. The MRI parameters and analysis, neurological scoring and behavioral testing are well described. WEAKNESSES: One reviewer felt that the lack of clarity about whether the transplanted cells will be purely neural stem cells or contaminated with hESC’s was a weakness. Also: • Specific Aim 3 is vague and unlikely to provide definitive data of neural networks as they suggest. • There are no details given about which brain metabolites they will study by MRS • The Abstract and Public Abstracts are identical and whole pieces from these are cut and pasted into the rationale and significance • There are problems with the number of cells and the volumes they plan to inject into the brain. Over 100,000 cells/ul leads to the cells clumping. These figures need to be reexamined. Another reviewer stated that: Specific aim 1: It is assumed from the description that all animals will undergo MRI on days 1 to 90 but there is no indication that the corresponding histological correlation between the MR findings (lesion size, cell migration, etc) and histological changes will be obtained. The number of animals in each group has not been indicated. The PI should include at least extra 3 animals for each time point to correlate with histological findings. The dose of Feridex is not mentioned. A section should be included to describe how the cells will be labeled and how the labeled cells will be analyzed to assess the viability, functional and differential capabilities. The PI should include another group of animals that will receive only unlabeled hESC derived neural stem cells (CCI+ unlabeled stem cells). This will give the effects of cells alone and can be determined by DWI and MRS analysis. Methods: The described number of cells and total injected volume is not practical. Considering the size of the rat brain, 250-500 µl volume can not be introduced either into ventricle or contra-lateral brain parenchyma. Considering the size of hESCs, it may not be possible to concentrate the cells at 900,000 per µl. What is the concentration of iron per cell? If 450x106 (based on 500 µl volume and 900,000 cell per 1 µl) labeled cells are introduced, there will be very large blooming effect (due to susceptibility of iron) which will impair the assessment of exact lesion volume and migration of cells. Feasibility: Description of the figures does not match with the corresponding figures. A legend for each illustration is desirable. DISCUSSION: A discussant offered that the proposal lacked detail about immunosuppression. Another discussion topic centered on whether the field would be advanced by the completion of this work; discussants considered it to be a replication of work that was conducted from ~1987-1990. One participant mentioned that toxicity was a concern, that hESCs are senstive to iron oxide and that no mention was made of this fact in the proposal. There was concern about the lack of experience of the applicant with behavioral testing, and how specific desired neurons would be obtained. No mention was made of neural markers to be used to characterize the population to be used for transplantation. The proposal was regarded as sloppily-written, with "cut and paste" sections evident.