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Belayev, Ludmila


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Belayev, Ludmila

  1. 1. 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 />
  2. 2. INTRODUCTION<br /><ul><li>Limited therapeutic options for patients after TBI
  3. 3. 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
  4. 4. 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
  5. 5. 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
  6. 6. A range of stimuli/conditions can increase the number of endogenous neural stem cells, including stroke, TBI, pregnancy, and exogenous pharmacologics
  7. 7. These newly generated cells can differentiate into a range of cells, including glia and new functioning neurons
  8. 8. 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 />
  9. 9. 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 />
  10. 10. 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 />
  11. 11.
  12. 12. 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 />
  13. 13. 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 />
  14. 14. Postural Reflex<br />Total Neurological Score<br />Placing Test<br />(Belayev et al., 1996)<br />
  15. 15. Neurological Outcome<br />Normal score = 0; maximal deficit =12<br />Both hCG+EPO groupsimproves neurological outcome<br />
  16. 16. Histopathology (3 weeks)<br />hCG+EPO (3 doses) reduces cortical contusion volume <br />
  17. 17. Cell Count (CA3 and Dentate Gyrus)<br />CA3<br />DG<br />hCG+EPO increases survival CA3 and DG neurons<br />
  18. 18. 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 />
  19. 19. T2WI<br />hCG+EPOreduces lesion cavities 3 weeks after TBI<br />
  20. 20. Ex vivo T2WI<br />hCG+EPOreduces ventricle size 3 weeks after TBI<br />
  21. 21. Possible Underlying Mechanisms?<br /><ul><li>Increases neurogenesis
  22. 22. Increases angiogenesis
  23. 23. Increases connectivity
  24. 24. Inhibits apoptosis
  25. 25. 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 />
  26. 26. Acknowledgements<br />Neuroscience Center of Excellence, LSUHSC, <br />New Orleans, LA<br />Loma Linda University,<br />Loma Linda, CA<br /><ul><li>Andy Obenaus
  27. 27. Pete Hayes
  28. 28. George Asberry</li></ul>Nicolas G. Bazan<br />Larissa Khoutorova<br />Stem Cell Therapeutics Corp<br />Calgary, Canada<br />Kristal Atkins<br />Tiffany Eady<br />