THE EFFECTS OF PERINATAL HYPOXIA/ISCHEMIA ON THE STEM CELLS AND PROGENITOR CELLS OF THE SVZ-DL IN VIVO
Open Access
- Author:
- Romanko, Michael John
- Graduate Program:
- Integrative Biosciences
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- July 23, 2004
- Committee Members:
- Stephen W Levison, Committee Chair/Co-Chair
Teresa L Wood, Committee Chair/Co-Chair
John Elliot Beard, Committee Member
Charles Palmer, Committee Member - Keywords:
- stem cells
progenitors
SVZ
caspase-3
calpains
stroke - Abstract:
- Hypoxic-ischemic (H/I) brain injury is a major cause of neurologic disability of the term and prematurely born infant. During the perinatal period the cells of the subventricular zone are actively dividing, migrating, and giving rise to the differentiated cells of the brain. The goals of this investigation were to: 1) define the time course of cell death within the dorsolateral subventricular zone (SVZDL); 2) determine the death mechanism of vulnerable cell types; 3) compare the vulnerability of stem and progenitor cells; and 4) determine a regenerative response. The SVZs of P6 rats were examined between 2 and 48h of recovery from H/I. Pyknotic nuclei and ISEL+ cells were present by 4h of recovery, peaked at 12h and persisted for at least 48h. The pyknotic cells corresponded to TUNEL+ cells. Many active-caspase-3 + cells were also present at 12h and comprised 1/3 of the total TUNEL+ population. Electron microscopy revealed that hybrid cell deaths predominated at 12h of recovery suggesting excitotoxic death. Consistent with excitotoxic death, m-calpain became activated as measured by Western blot analysis of fodrin cleavage, as early as 4h of recovery and persisted strongly to 48h of recovery. Calpain can activate caspase-3 and a Western blot analysis of caspase-3 showed that active caspase-3 first became detectable at 8h of recovery, and peaked at 24h following the insult. This corresponded to cell counts of active caspase-3+ cells which showed a near 5 fold increase at 24h. Few dying cells were observed in the medial SVZ, where putative stem cells reside, and no nestin+ medial SVZ cells showed caspase-3 activation. Active-caspase- 3+/PSA-NCAM+ progenitors were prominent in the lateral SVZ. These data suggest that early progenitors are vulnerable to H/I, whereas neural stem cells are resilient. Proliferation and expansion of the stem population occurred in response to the insult. By 48h of recovery nearly twice as many neurospheres could be generated from ipsilateral ischemic SVZ. Double immunoflourescence showed a 2 fold increase in the number of dividing PCNA+/nestin+ cells in the most medial region. It is likely that the demise of these progenitors may contribute to cerebral dysgenesis after perinatal insults.