Delayed post-treatment of neonatal hypoxic-ischemic striatal injury with bone marrow-derived mesenchymal stem cells: An immunohistochemical/stereological and behavioural study

Abstract

Hypoxia/ischemia encephalopathy is a major contributor to the development of acute neonatal neurodevelopmental deficits, such as cerebral palsy (CP). One of the major sites afflicted by neonatal H/I injury is the striatum of the basal ganglia. Medium- spiny neurons (MSNs) comprise >97% of striatal neurons and play an important role in voluntary motor control. Damage to the striatum following neonatal H/I injury results in selective death of MSNs, and is correlated with the motor dysfunction that afflicts CP children. It is therefore clinically relevant to target the neuroprotection or neurorestoration of MSNs. Moderate hypothermia, a neuroprotective treatment that is currently used clinically, must be administered within 6 hours post-injury to be effective. A neurorestorative treatment that extends this window for therapeutic intervention would be highly beneficial. The neurorestorative potential of delayed treatment with bone marrow-derived mesenchymal stem cells (MSCs) was investigated in this thesis. Experimental procedures involved the exposure of postnatal day (PN) 7 male rats to H/I injury. Animals were then treated with a delayed subcutaneous high dose (HD, 7.70 x 105-1.43 x 106) of MSCs or diluent on PN14. Immunohistochemical/stereological and long-term behavioural studies were performed to determine if any effect was observed. This study first aimed to histologically confirm the findings of Alwakeel (2011) that demonstrated the restoration of the absolute number of striatal MSNs following a delayed HD MSC- treatment. A specific marker of MSNs, DARPP-32, was used. Secondly, this study investigated the neurogenic subventricular zone (SVZ) as the source of progenitor cells from which newly generated MSNs are derived. This involved double-immunolabelling with bromodeoxyuridine (BrdU), a progenitor cell marker, and DARPP-32. The Cavalieri and unfolding methods were used to stereologically quantify the absolute number of DARPP-32 and BrdU/DARPP-32-positive neurons within the striatum. MSC-treatment significantly increased the absolute number of DARPP-32-positive and BrdU/DARPP-32- positive striatal neurons. These results confirm the neurorestorative potential of MSCs. This study is the first to provide evidence of the SVZ as the neurogenic source of replacement striatal MSNs following delayed MSC-treatment of neonatal H/I injury. Behavioural testing investigated whether MSC-treatment was capable of long- term attenuation of motor function deficits resulting from neonatal hypoxia/ischemia. Repeated cylinder testing was performed on PN21, PN35, PN88-90, and PN162-164. Staircase testing and foot-fault testing were performed between PN61-86, and on PN103-105, respectively. Cylinder testing on PN35 and PN90, as well as staircase testing, demonstrated significant motor deficits of H/I diluent-treated animals compared to the uninjured control cohort. Significant motor deficits were not demonstrated between H/I MSC-treated and uninjured control cohorts. These results may be suggestive of motor improvement of MSC-treated animals. The foot-fault test did not demonstrate significant differences in motor performance between cohorts. The therapeutic potential of bone-marrow derived MSCs for the delayed treatment of neonatal hypoxia/ischemia is therefore supported by the findings of this study. The endogenous regenerative processes of the neonatal brain, through trophic enrichment provided by MSC-treatment, may be harnessed to drive the neurorestoration of MSNs. Further investigation should be actively pursued to ready MSCs for clinical use for the treatment of children with CP.