Abstract
Perinatal hypoxia-ischemia (HI), resulting from a sudden decrease in the levels of blood and oxygen to the neonatal brain, is a major cause of striatal injury. Infants suffering from a HI attack have an increased risk of postnatal mortality and permanent neuropsychologic deficits. This signifies a need for treatments capable of reducing the disease burden of HI. Stem cell-based therapies have been extensively examined in perinatal HI. Among all lineages of stem cells evaluated, the mesenchymal stem cells (MSCs), particularly adipose-derived MSCs (AD-MSCs), have yielded encouraging results for future therapies of HI.
The present study first aimed to evaluate the efficacy of AD-MSCs in attenuating the inflammatory response in the dorsolateral subventricular zone (dlSVZ) and striatum of neonatal rats exposed to HI at term-equivalent. It was hypothesised that the primary neurorestorative effect of AD-MSCs emerges from their anti-inflammatory effects. This was hypothesised to be achieved by reducing the number of resident microglial cells in the dlSVZ and caudate-putamen (CPu). Postnatal day (PN) 7/8 rat pups were exposed to HI and assigned to either (1) treatment with diluent (DPBS containing 0.5% bovine serum albumin, BSA) or (2) double treatment with AD-MSCs in diluent on PN14/15 and PN16/17. The rats were perfused on PN17/18. Serial coronal 5 µm sections were immunohistochemically stained for anti-ionised calcium-binding adaptor protein 1 (IBA-1) positive microglial cells in the CPu and dlSVZ. The total reference volume, density of microglial cells, and the absolute number of microglial cells were determined per structure using stereological methods. There was a statistically significant decrease in the absolute number of IBA-1 positive microglial cells in the CPu of AD-MSC treated pups compared to their diluent counterparts. No significant decrease in the absolute number of IBA-1 immunolabelled microglia cells in the dlSVZ was found between AD-MSC-treated and diluent-treated HI pups. However, AD-MSC-treated pups had a statistically significant lower density of IBA-1 positive microglial cells in their dlSVZ compared to their diluent-treated pairs. Taken together, the results of the current study indicate that double treatment with AD-MSCs has an anti-inflammatory action in the CPu and dlSVZ of neonatal HI pups.
Aghoghovwia (2018; see Figure 1.10) has previously shown that there no increase in the absolute number (N) of striatal dopamine- and cAMP-regulated phosphoprotein-32 (DARPP-32)-positive spiny projection neurons (SPNs) in HI rat pups treated with a single dose of AD-MSCs compared to the HI-diluent-treated littermates (n=4). This led to the current hypothesis, that by increasing the statistical power by analysing more HI rat pups, a single delayed treatment with AD-MCS will restore the number of striatal SPNs to normal levels. PN7 rat pups were exposed to HI and allocated to either treatment with: (1) diluent, (2) a single administration of AD-MSCs in diluent (AD-MSCsx1), (3) double administration of AD-MSCs in diluent (AD-MSCsx2), or (4) not subjected to HI injury but treated with diluent (i.e. these were normal control pups). All pups were also injected intraperitoneally with 5′-bromo-2′-deoxyuridine (BrdU), every 24 hours, on PN15, PN16 and PN17, to track the migration of cells from the SVZ, and their differentiation into SPNs, in the adjacent injured striatum. Pups were perfused on PN21. Following double-immunostaining for DARPP-32 and BrdU, the absolute number of DARPP-32 and double-labelled DARPP-32/BrdU striatal SPNs was stereologically measured. The present study revealed, as its major finding, a statistically significant increase in the absolute number of DARPP-32-positive SPNs following AD-MSCsx2-, but not AD-MSCsx1-, treatment compared to their diluent-treated littermates. These results provide evidence that two delayed administrations of AD-MSCs are needed to generate a therapeutic benefit.