Gracee Agrawal

Efficient differentiation of human embryonic stem cells into oligodendrocyte progenitors for application in a rat contusion model of spinal cord injury

ABSTRACT This study utilized a contusion model of spinal cord injury (SCI) in rats using the standardized NYU-MASCIS impactor, after which oligodendrocyte progenitor cells (OPCs) derived from human embryonic stem cell (ESC) were transplanted into the spinal cord to study their survival and migration route toward the areas of injury. One critical aspect of successful cell-based SCI therapy is the time of injection following injury. OPCs were injected at two clinically relevant times when most damage occurs to the surrounding tissue, 3 and 24 hours following injury. Migration and survivability after eight days was measured postmortem. In-vitro immunofluorescence revealed that most ESC-derived OPCs expressed oligodendrocyte markers, including CNPase, GalC, Olig1, O4, and O1. Results showed that OPCs survived when injected at the center of injury and migrated away from the injection sites after one week. Histological sections revealed integration of ESC-derived OPCs into the spinal cord with contusion injury without disruption to the parenchyma. Cells survived for a minimum of eight days after injury, without tumor or cyst formation. The extent of injury and effect of early cell transplant was measured using behavioral and electrophysiological assessments which demonstrated increased neurological responses in rats transplanted with OPCs compared to controls.

Characterization of graded multicenter animal spinal cord injury study contusion spinal cord injury using somatosensory-evoked potentials.

Study Design. Electrophysiological analysis using somatosensory-evoked potentials (SEPs) and behavioral assessment using Basso, Beattie, Bresnahan (BBB) scale were compared over time for graded Multicenter Animal Spinal Cord Injury Study (MASCIS) contusion spinal cord injury (SCI). Objective. To study the SEP responses across different contusion injury severities and to compare them with BBB scores. Summary of Background Data. For any SCI therapy evaluation, it is important to accurately and objectively standardize the injury model. The graded MASCIS contusion injuries on dorsal spine have been standardized using BBB, which is subjective and prone to human errors. Furthermore, dorsal pathway disruption does not always produce locomotor deficits. SEP monitoring provides an advantage of providing a reliable and objective assessment of the functional integrity of dorsal sensory pathways. Methods. Four groups of Fischer rats received contusion at T8 using New York University (NYU)-MASCIS impactor from impact heights of 6.25 mm (mild), 12.5 mm (moderate), 25 mm (severe), or 50 mm (very severe). The control group underwent laminectomy only. SEP and BBB recordings were performed once before injury, and then weekly for up to 7 weeks. Results. Graded levels of injury produced concomitant attenuations in hindlimb SEP amplitudes. Following injury, 25 and 50 mm groups together differed significantly from 12.5 and 6.25 mm groups (P < 0.01). From week 5, differences between 12.5 and 6.25 mm groups also became apparent (P < 0.01), which showed significant electrophysiological improvement. However, no significant differences were observed between 25 and 50 mm groups, which showed negligible electrophysiological recovery. Although comparable differences between different groups were also detected by BBB after injury (P < 0.001), BBB was less sensitive in detecting any improvement in 6.25 and 12.5 mm groups. Conclusion. SEP amplitudes and BBB scores decrease corresponding to increase in injury severity, however, these show different temporal patterns of recovery. These results demonstrate the utility of SEPs in conjunction with BBB, to monitor therapeutic interventions in SCI research.

Human Glial-Restricted Progenitors Survive, Proliferate, and Preserve Electrophysiological Function in Rats with Focal Inflammatory Spinal Cord Demyelination

Transplantation of glial progenitor cells results in transplant‐derived myelination and improved function in rodents with genetic dysmyelination or chemical demyelination. However, glial cell transplantation in adult CNS inflammatory demyelinating models has not been well studied. Here we transplanted human glial‐restricted progenitor (hGRP) cells into the spinal cord of adult rats with inflammatory demyelination, and monitored cell fate in chemically immunosuppressed animals. We found that hGRPs migrate extensively, expand within inflammatory spinal cord lesions, do not form tumors, and adopt a mature glial phenotype, albeit at a low rate. Human GRP‐transplanted rats, but not controls, exhibited preserved electrophysiological conduction across the spinal cord, though no differences in behavioral improvement were noted between the two groups. Although these hGRPs myelinated extensively after implantation into neonatal shiverer mouse brain, only marginal remyelination was observed in the inflammatory spinal cord demyelination model. The low rate of transplant‐derived myelination in adult rat spinal cord may reflect host age, species, transplant environment/location, and/or immune suppression regime differences. We conclude that hGRPs have the capacity to myelinate dysmyelinated neonatal rodent brain and preserve conduction in the inflammatory demyelinated adult rodent spinal cord. The latter benefit is likely dependent on trophic support and suggests further exploration of potential of glial progenitors in animal models of chronic inflammatory demyelination.

Evoked potential versus behavior to detect minor insult to the spinal cord in a rat model.

Reliable outcome measurement is needed for spinal cord injury research to critically evaluate the severity of injury and recovery thereafter. However, such measurements can sometimes be affected by minor, injury to the spinal cord during surgical procedures, including laminectomy. The open-field Basso, Beattie and Bresnahan (BBB) behavior motor scores are subjective and prone to human error. We investigated somatosensory evoked potential (SEP) as an electrophysiological measure to assess the integrity of the spinal cord after injury. In our experiment, control rats with a minor unintentional spinal cord insult during laminectomy showed a decrease in SEP amplitude by 16% to 18%, which recovered in around 7 days. However, there was no change in the BBB scores for the same animals over the same period. This highlights the sensitivity of SEP to minor insult as compared to BBB. These differences may be beneficial in accurate evaluation of the severity and progression of spinal cord injury, and subsequent recovery.

Slope analysis of somatosensory evoked potentials in spinal cord injury for detecting contusion injury and focal demyelination

In spinal cord injury (SCI) research there is a need for reliable measures to determine the extent of injury and assess progress due to natural recovery, drug therapy, surgical intervention or rehabilitation. Somatosensory evoked potentials (SEP) can be used to quantitatively examine the functionality of the ascending sensory pathways in the spinal cord. A reduction of more than 50% in peak amplitude or an increase of more than 10% in latency are threshold indicators of injury. However, in the context of injury, SEP peaks are often obscured by noise. We have developed a new technique to investigate the morphology of the SEP waveform, rather than focusing on a small number of peaks. In this study, we compare SEP signals before and after SCI using two rat models: a contusion injury model and a focal experimental autoimmune encephalomyelitis model. Based on mean slope changes over the signal, we were able to effectively differentiate pre-injury and post-injury SEP values with high levels of sensitivity (83.3%) and specificity (79.2%).

Effect of MOG sensitization on somatosensory evoked potential in Lewis rats.

Myelin oligodendrocyte glycoprotein (MOG) is commonly used as an immunogen to induce an immune response against endogenous myelin, thereby modeling multiple sclerosis in rodents. When MOG is combined with complete Freunds adjuvant (CFA), multifocal, multiphasic disease ensues; whereas when MOG is combined with incomplete Freunds adjuvant (IFA), clinical disease is usually absent. MOG-IFA immunized animals can be induced to have neurological disease after intraspinal injections of cytokines and ethidium bromide (EtBr). In this study, we investigated whether MOG-IFA immunized rats exhibited subclinical injury as defined by somatosensory evoked potential (SEP) recordings. The titration of anti-MOG-125 antibodies showed robust peripheral mounting of immune response against myelin in MOG-immunized rats. However the SEP measures showed no significant change over time. Upon injecting cytokine-EtBr in the spinal cord after MOG sensitization, the SEP recordings showed reduced amplitude and prolonged latency, suggestive of axonal injury and demyelination in the dorsal column, respectively. These findings were later confirmed using T2-weighted MRI and histological hematoxylin-eosin stain of the spinal cord. This report establishes that MOG-IFA immunization alone does not alter neuronal conduction in SEP-related neural-pathways and that longitudinal in-vivo SEP recordings provide a sensitive read-out for focal myelitis (MOG-IFA and intraspinal cytokine-EtBr) in rats.

Spinal Cord Injury Detection and Monitoring Using Spectral Coherence

In this paper, spectral coherence (SC) is used to study the somatosensory evoked potential (SEP) signals in rodent model before and after spinal cord injury (SCI). The SC technique is complemented with the Basso, Beattie, and Bresnahan (BBB) behavior analysis method to help us assess the status of the motor recovery. SC can be used to follow the effects of SCI without any preinjury baseline information. In this study, adult female Fischer rats received contusion injury at T8 level with varying impact heights using the standard New York University impactor. The results show that the average SC between forelimb and hindlimb SEP signals before injury was relatively high ( ges0.7). Following injury, the SC between the forelimb and hindlimb SEP signals dropped to various levels (les0.7) corresponding to the severity of SCI. The SC analysis gave normalized quantifiable results for the evaluation of SCI and recovery thereafter using the forelimb signals as an effective control, without the need of any baseline data. This technique solves the problems associated with the commonly used time-domain analysis like the need of a trained neurophysiologist to interpret the data and the need for baseline data. We believe that both SC and BBB may provide a comprehensive and complementary picture of the health status of the spinal cord after injury. The presented method is applicable to SCIs not affecting the forelimb SEP signals.

Evoked potential and behavioral outcomes for experimental autoimmune encephalomyelitis in Lewis rats

A reliable outcome measurement is needed to assess the effects of experimental lesions in the rat spinal cord as well as to assess the benefits of therapies designed to modulate them. The Basso, Beattie, and Bresnahan (BBB) behavioral scores can be indicative of the functionality in motor pathways. However, since lesions are often induced in the more accessible dorsal parts associated with the sensory pathways, the BBB scores may not be ideal measure of the disability. We propose somatosensory evoked potential (SEP) as a complementary measure to assess the integrity of sensory pathways. We used the focal experimental autoimmune encephalomyelitis (EAE) model, in which focal demyelinating lesions were induced by injecting cytokine-ethidium bromide into dorsal white matter after MOG-IFA immunization. Both the SEP and BBB measures reflected injury; however, the SEP was uniformly and consistently altered after the injury whereas the BBB varied widely. The results suggest that the SEP measures are more sensitive and reliable markers of focal spinal cord demyelination compared to the behavioral measures like the BBB score.

A comparative study of recording procedures for motor evoked potential signals

Motor evoked potential (MEP) signals serve as an objective measure of the functional integrity of motor pathways in the spinal cord. Hence, they provide a reliable assessment of the extent of spinal cord injury (SCI). There are two methods currently being used for serial MEP recordings in rats: a low-frequency and a high-frequency method. In this paper, we compared the two methods and determined the better method for MEP recordings. We also compared the effect of two anesthetic agents — inhalational isoflurane and intraperitoneal ketamine — on the MEP signals. We found that under ketamine anesthesia, low-frequency stimulation led to more consistent results, while high-frequency stimulation required greater stimulation intensity and was prone to unwanted side-effects including excessive head twitches. We further found that isoflurane anesthesia severely depressed the MEP response for both low-frequency and high-frequency stimulation which rendered the resulting signal unusable.

A novel shape analysis technique for somatosensory evoked potentials

Somatosensory evoked potentials (SEP) have been shown to be an important electrophysiological measure to assess the integrity of the spinal cord. However the peaks in the SEP waveform are often undetectable due to low signal-to-noise (SNR) ratio. Sometimes they also become indistinct during injury when the SEP flattens. Hence time-domain analysis methods are often subject to errors, and need human-expert intervention. In this paper, we propose a new technique for analyzing the shape of the evoked potentials, in which slope information is obtained for the entire signal in specific time bins. Apart from solving the problems associated with present methods, this technique has an added advantage of analyzing the SEP signal as a whole rather than simply a few peaks. The efficacy of this technique was investigated on SEP signals recorded from 12 rats before and after contusion spinal cord injury at thoracic vertebra T8. The statistical analysis results revealed significant effect of injury to hindlimbs, whereas almost none to forelimbs. Thus, the results show high potential of this technique to differentiate between normal and injured spinal cord.