Perinatal hypoxia-ischaemia is a major cause of disability, including cerebral palsy, yet a neuroprotectant which fully protects the brain remains elusive. Following a hypoxic-ischaemic insult, striatal medium-spiny neurons and hippocampal CA1 neurons are vulnerable to a complex cascade of neurotoxic events. This cascade includes energy failure, a massive release of glutamate, the formation of free radicals and caspase activation. The overall aim of this thesis was to assess the efficacy of three potential neuroprotective strategies that target this cascade from different directions. Short-term, and where appropriate, long-term, neuroprotection was investigated. The first treatment strategy aimed to suppress the generation of free radicals through treatment with the potent free radical spin trap, N-tertbutyl-(2-sulphophenyl)-nitrone (S-PBN). The second compound tested was the caspase-3 inhibitor, minocycline. Finally, the third treatment strategy combined a series of S-PBN injections with 6 hours of moderate hypothermia immediately after hypoxia-ischaemia. Hypothermia is suggested to slow the rate of the neurotoxic cascade, thus potentially allowing other neuroprotective agents greater efficacy.
Using an adaptation of the Rice et al. (1981) model, hypoxia-ischaemia was induced on postnatal day (PN) 8 in the right cerebral hemisphere. For the short-term studies, the rats were perfused at 14 days-of-age. The brains were dissected out and embedded in Technovit. Forty [mu]m serial sections were cut through the right striatum and hippocampus. The total number of medium-spiny neurons in the striatum and where appropriate, the total number of neurons in the hippocampal CA1 pyramidal layer, were stereologically determined using the optical disector/Cavalieri method.
For the long-term study, fine motor control was assessed in half of the animals through the staircase test from 9-11 weeks-of-age. Neuroprotection was assessed in the remaining animals. All animals were sacrificed at 12 weeks-of-age. The total number of striatal medium-spiny neurons was stereologically determined in the non-behavioural animals as described above.
A series of seven injections of S-PBN (100mg/kg) did not offer statistically significant neuroprotection to the striatum at one week after perinatal hypoxia-ischaemia. Similarly, a single injection of minocycline (45mg/kg) immediately after the insult did not offer significant neuroprotection to the striatum nor the CA1 region of the hippocampus at this early time-point. In contrast, when the series of S-PBN injections was combined with 6 hours of moderate hypothermia post-hypoxia-ischaemia, sterelogical analysis revealed significant neuroprotection of the striatal medium-spiny neurons to normal levels at one week after the injury. No significant neuroprotection was seen in the CA1 region of the same animals.
To assess whether this impressive striatal neuroprotection was long-lasting and whether it represented functional rescue, the final experiment in this thesis investigated rat pups at 12 weeks-of-age after exposure to hypoxia-ischaemia at PN8. Treatment with S-PBN/hypothermia offered persistent neuroprotection of striatal medium-spiny neurons and preservation of fine motor skills compared to diluent-normothermia-treated controls. The long-term behavioural outcomes were compared with normal, uninjured controls and the total number of medium-spiny neurons was compared with normal numbers from the literature. These comparisons revealed that the histological and functional integrity of the striatum was rescued to normal levels.
This is the first study to identify a treatment strategy that offers complete and long-lasting preservation of striatal neuronal numbers, by accurate and unbiased stereological methods, paired with persistent preservation of fine motor control following perinatal hypoxia-ischaemia.
| Identifer | oai:union.ndltd.org:ADTP/217479 |
| Date | January 2005 |
| Creators | Hobbs, Catherine E., n/a |
| Publisher | University of Otago. Department of Anatomy & Structural Biology |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | http://policy01.otago.ac.nz/policies/FMPro?-db=policies.fm&-format=viewpolicy.html&-lay=viewpolicy&-sortfield=Title&Type=Academic&-recid=33025&-find), Copyright Catherine E. Hobbs |
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