In the next study we used MCAo for 2 h and we used hCG instead of Epidermal growth factor because there is more human experience with hCG, and both are thought to be endogenous stem cell mitogens.hCG+EPO significantly improved behavioral score and reduced lesion volume during 3 month survival period.
This combination of hCG + EPO was safe, when was given to the patients during 24-48h after stroke onset.
Both groups treated with hCG-EPO Improved Neurological Outcome started on day 14 and continue until day 21.
Normal cell count was increased in in the right and left side of CA3 sector of hippocampus in both hCG/EPO groups and hCG alone. Treatment with 3 doses hCG/EPO significantly increased normal cell count in the dentate gyrus.
TBI produced a lesion cavity in the injured cortex. Compared with vehicle groups, hCG-EPO treatment reduced lesion cavity at 3 weeks after injury.
Total impact damage was reduced by 3 doses hCG-EPOtreatment, but it was not statistically significant.Ventricular dilatation was present in the saline treated rats. In contrast, ventricle size in both hCG-EPO treated group was close to normal. Also treatment with Sal-EPO reduced ventricle size as well.
A NOVEL THERAPEUTIC STRATEGY FOR TRAUMATIC BRAIN INJURY <br />Ludmila Belayev, MD<br />David Zygun, MD<br />Kristal D. Atkins, MS<br />Larissa Khoutorova, BS <br />Tiffany N. Eady, BS<br />Allen W. Davidoff, PhD<br />Nicolas G. Bazan, MD, PhD<br />LSUHSC, New Orleans, LA<br />Stem Cell Therapeutics, Calgary, Alberta, Canada<br />Alberta Health Services-Foothills Hospital, Calgary, Alberta, Canada<br />
INTRODUCTION<br /><ul><li>Limited therapeutic options for patients after TBI
A number of studies have suggested that modification of post-injury events through pharmacologic intervention can promote functional recovery in both a variety of animal models and clinical CNS injury
Recently, attention has focused on potential therapeutic agents that enhance endogenous neuroplasticity, including neurogenesis after brain injury, with a final goal of improving functional outcome
Such interventions to promote repair rather than limit initial injury - “restorative therapies” - might in part be mediated through endogenous neural stem cells</li></li></ul><li>Endogenous Neural Stem Cells<br /><ul><li>The mature mammalian brain continually generates new neurons in the regions of subventricular zone (SVZ) and the dentate gyrus (DG) of the hippocampus throughout life
A range of stimuli/conditions can increase the number of endogenous neural stem cells, including stroke, TBI, pregnancy, and exogenous pharmacologics
These newly generated cells can differentiate into a range of cells, including glia and new functioning neurons
This injury-enhanced cell proliferation has been linked to improved cognitive function following brain injury</li></li></ul><li>Hypothesis<br />We use two approved and clinically well-defined drugs:<br />Human Chorionic Gonadotropin (hCG) is the first drug administered in the regimen and aims to increase the number of neural stem cells (NSCs) located in the brain after TBI. <br />Erythropoietin (EPO)is the second drug administered in the regimen and aims to promote the differentiation of these newly formed NSCs into new neurons. <br />New neurons thus formed are anticipated to provide benefits through the replacement of brain cells that were lost or damaged by the TBI.<br />
Vehicle<br />EGF+EPO<br />Hypotheses Supported by Prior Work<br />Pial strip injury model in rats<br />hCG (at 24h) daily over 7 days<br />BrdU positive cells (SVZ)<br />Pial strip injury model in rats<br />Epidermal growth factor (EGF), an agent to induce proliferation (ICV, 7days)<br />Erythropoietin (EPO), an agent to support survival of newly formed progenitors as well as induce differentiation (IV, 7days) <br />hCG<br />hCG<br />hCG (at 24h) on days 1, 3 and 5<br />Doublecortin(neurons, SVZ)<br />hCG<br />EGF + EPO promotes substantial regeneration of damaged cerebral cortex and improves behavioral deficit<br />(Kolb et al, 2007)<br />
Hypotheses Supported by Prior Work<br />Long Evans rats<br />MCAo, 2h<br />hCG (450 IU/day, IM, on day 1, 3 and 5)<br />EPO (1440 IU/day , IV on days 6,7,8 <br />Histopathology (3 months)<br />Total Neurological Score<br />(Belayev et al, 2009)<br />hCG-EPO: improves motor function and reduces lesion volumes after MCAo<br />
Human Chorionic Gonadotropin (hCG)<br />Erythropoietin (EPO)<br />hCG is a glycoprotein hormone, produced in pregnancy. It is in the same growth factor family as nerve growth factor<br />Serum hCG levels: normal < 3 IU/L, pregnancy> 100,000 IU/L<br />Receptor normally present in adult and fetal rat brains, particularly in the hippocampus<br />Crosses the BBB (Lukacs et al, 95)<br />Neurons cultured with hCG show dose-dependent increase in neurite outgrowth & decrease apoptosis (al-Hader et al, 97)<br />Improves behavioral outcome, tissue spanning transection, and neurophysiology after SCI in rats<br />Some effects mediated by PGE2, PGD2, 5-LO (Toth 94; Zhang 99)<br />EPO is a glycoprotein hormone that controls red blood cell production. <br />It is used in treating anemia resulting from kidney disease and from the treatment of cancer<br />EPO crosses BBB (Kaushansky, 2006)<br />Erythropoietin and EPOR are expressed in neuronal, astrocytic, and endothelial cells (Brines, 2000)<br />Promotes differentiation of adult neural stem cells into neurons (Shingo et al, 2001)<br />Important to adult neurogenesis and neuronal migration (Tsai et al, 2006)<br />Anti-apoptotic (Digicaylioglu 01) and pro-angiogenic (Xiong et al, 2011) <br />
Experimental Protocol<br />Animals: Male Sprague Dawley rats (285-360 g) <br />Anesthesia: Isoflurane/nitrous oxide/oxygen <br />Physiological Monitoring: Rectal and cranial temperatures, arterial blood gases (pO2 , pCO2 ) and pH, plasma glucose, hematocrit.<br />Animal Model: Fluid Percussion Injury (severe, 7-8atm) in right parieto-occipital area. <br />Behavioral Testing <br />Days<br />Time<br />-24h<br />Time<br />0h<br />14<br />6 7 8<br />21<br />1 2 3 4 5<br />FPI<br />Preparation<br />for FPI<br />hCG<br />300 IU/kg<br />IM<br />EPO<br />1440 IU/kg<br />IV<br />Experimental groups:<br />hCG-3 doses (1,3,5d) followed by EPO <br />hCG-5 doses (1-5d) followed by EPO <br />hCG-3 doses followed by Saline<br />Saline followed by EPO <br />Saline followed by Saline<br />Ex vivo MRI<br />Histopathology<br />
Cell Count (CA3 and Dentate Gyrus)<br />CA3<br />DG<br />hCG+EPO increases survival CA3 and DG neurons<br />
Magnetic Resonance Imaging (MRI)<br />Ex vivo imaging on a Bruker Advance 11.7T MRI<br />11.7T Bruker Advance 30cm horizontal bore instrument equipped with an 89 mm (ID) quadrature receiver coil (BrukerBiospin, Billerica, MA). <br />T2WI sequence (2395.9 ms/10.2ms of TR/TE, 2 cm of field of view (FOV), 20 slices, 1mm slice thickness,256x256 matrix)<br />DTI sequence in seven directions with two b-values (b0 = 43.340 and b= 2013.464 s/mm^2), (2000ms/15.1ms TR/TE, 2cm FOV, 20 slices, 1mm slice thickness)<br />Analysis:<br />T2WI<br />Region of interest analysis (Cheshire software)<br />Lesion volume<br />Brain volume and ventricle size<br />
T2WI<br />hCG+EPOreduces lesion cavities 3 weeks after TBI<br />
Ex vivo T2WI<br />hCG+EPOreduces ventricle size 3 weeks after TBI<br />
Possible Underlying Mechanisms?<br /><ul><li>Increases neurogenesis
Inhibits inflammatory response</li></li></ul><li>CONCLUSIONS<br />The two-drug approach, initiated 24h after TBI, showed some advantages compared to the one-drug approach and confers<br />Behavioral improvement<br />Reduction of lesion volume<br />Reduction of hippocampal cell loss<br />Extensive human experience exists for both hCG and EPO, suggesting high potential for successful translation into a clinical treatment for TBI. <br />
Acknowledgements<br />Neuroscience Center of Excellence, LSUHSC, <br />New Orleans, LA<br />Loma Linda University,<br />Loma Linda, CA<br /><ul><li>Andy Obenaus