Oxygen-glucose deprivation modelling and overnight incubation ofacute hippocampal slices in vitro
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- Tez No: 758757
- Danışmanlar: PROF. DR. ANDREAS DRAGUHN, DR. ANA M. M. OLİVEİRA
- Tez Türü: Yüksek Lisans
- Konular: Biyoloji, Biyomühendislik, Biology, Bioengineering
- Anahtar Kelimeler: Belirtilmemiş.
- Yıl: 2021
- Dil: İngilizce
- Üniversite: Ruprecht-Karls-Unıversıtat-Heıdelberg
- Enstitü: Yurtdışı Enstitü
- Ana Bilim Dalı: Belirtilmemiş.
- Bilim Dalı: Belirtilmemiş.
- Sayfa Sayısı: Belirtilmemiş.
Özet
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Özet (Çeviri)
During hypoxia-ischemia, as occurring in ischemic stroke, neurons are deprived of oxygen and glucose, which within minutes leads to neuronal network dysfunction. Hippocampal pyramidal cells (PYR) and fast-spiking interneurons (FSI) are particularly vulnerable to oxygen-glucose deprivation (OGD). Previous studies on tissue from young adult mice have shown that the dysfunction of FSI may contribute to impaired network recovery following OGD. However, currently no data exists on aged mice, although this group is from particular interest as stroke primarily affects the elderly. The aims of this thesis are 1) to close this knowledge gap by studying the recovery of neuronal function from acute hypoxia-ischemia on cellular and network levels in acute brain slices from aged mice, 2) to compare the effects of mild vs. severe OGD on axonal morphology and 3) to extend the experimental time window in acute brain slices. OGD was modelled in in acute slices from 75- to 104-week-old mice by switching the perfusion and gas to an oxygen- and glucose-free medium, until a spreading depression (SD) was induced. SD was detected extracellularly by a large negative potential shift and a transient cessation of neuronal firing. Tetrode recordings were performed in the hippocampal CA1 and CA3 regions during OGD, until 60 min and after four hours of recovery to measure single-cell and neuronal network activity. The hippocampus in our preparation spontaneously entrains its innate form of network activity, the sharp wave-ripple (SPW-R) oscillations, which are crucial for spatial and episodic memory formation. After one hour, and even more so after four hours of recovery SPW-R frequency was reduced in both CA1 and CA3 regions. At one hour of recovery FSI showed a reduced firing rate, while PYR spiking was not significantly altered. After four hours, FSI firing remained reduced compared to control in both regions, while PYR firing increased. Overall, unit firing of the CA1 region was less coupled to SPW-R compared to control. As changes in the excitatory-inhibitory balance have been shown to be accompanied by changes in axon initial segment (AIS) morphology, slices were stained for β4-spectrin, a marker of AIS, after experiments. However, no changes in AIS length nor numbers were detected after fourhour recovery from mild OGD. In contrast, a more severe OGD for 20 min severely diminished AIS numbers and length in a separate set of experiments. In a next step, irradiation of artificial cerebrospinal fluid (ACSF) with ultraviolet (UV) light and addition of the antibiotics penicillin and streptomycin was used to extend the viability of 4 acute slices. After 21 hours experimental slices still had SPW-R activity in their CA3 region but not in their CA1 region and the control slices, which were perfused with normal ACSF without the added UV light nor antibiotics, showed no SPW-R activity in both regions. No difference in AIS length nor numbers were detected between experiment and control slices for the CA1 region. Together, these results in tissue from aged mice show similar effects of OGD on FSI and SPWR activity as shown in young mice in a previous study from our group and support the hypothesis of FSI dysfunction following mild hypoxia-ischemia. Furthermore, they suggest an increasing inhibitory deficit over time. While AIS morphology is unaltered after mild OGD, we show that AIS break down after severe OGD, which may be an important pathomechanism of ischemic neuronal damage. Furthermore, we prove that it is feasible to maintain acute slices expressing spontaneous SPW-R activity after 21 hours of incubation, which significantly widens the time window for experiments and enables the study of processes like delayed apoptosis and long-lasting changes in network activity.
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