Elsevier

Brain Research Bulletin

Volume 135, October 2017, Pages 163-169
Brain Research Bulletin

Technical report
A novel method for oxygen glucose deprivation model in organotypic spinal cord slices

https://doi.org/10.1016/j.brainresbull.2017.10.010Get rights and content

Highlights

  • This study established a model to closely mimic spinal cord hypoxic-ischemic injury.

  • OGD model is obtained from long-term cultured organotypic spinal cord slices.

  • For OGD model, 60 min was the optimal time.

Abstract

This study aimed to establish a model to closely mimic spinal cord hypoxic-ischemic injury with high production and high reproducibility. Fourteen-day cultured organotypic spinal cord slices were divided into 4 groups: control (Ctrl), oxygen glucose deprived for 30 min (OGD 30 min), OGD 60 min, and OGD 120 min. The Ctrl slices were incubated with 1 ml propidium iodide (PI) solution (5 μg/ml) for 30 min. The OGD groups were incubated with 1 ml glucose-free DMEM/F12 medium and 5 μl PI solution (1 mg/ml) for 30 min, 60 min and 120 min, respectively. Positive control slice was fixed by 4% paraformaldehyde for 20 min. The culture medium in each group was then collected and the Lactate Dehydrogenase (LDH) level in the medium was tested using Multi-Analyte ELISArray kits. Structure and refraction of the spinal cord slices were observed by light microscope. Fluorescence intensity of PI was examined by fluorescence microscopy and was tested by IPP Software. Morphology of astrocytes was observed by immunofluorescence histochemistry. Caspase 3 and caspase 3 active in different groups were tested by Western blot. In the OGD groups, the refraction of spinal cord slices decreased and the structure was unclear. The changes of refraction and structure in the OGD 120 min group were similar to that in the positive control slice. Astrocyte morphology changed significantly. With the increase of OGD time, processes became thick and twisted, and nuclear condensations became more apparent. Obvious changes in morphology were observed in the OGD 60 min group, and normal morphology disappeared in the OGD 120 min group. Fluorescence intensity of PI increased along with the extension of OGD time. The difference was significant between 30 min and 60 min, but not significant between 60 min and 120 min. The intensity at OGD 120 min was close to that in the positive control. Compare with the Ctrl group, the OGD groups had significantly higher LDH levels and caspase 3 active/caspase 3 ratios. The values increased with the extension of OGD time and reached peak at 120 min. The increase was significant between 30 min and 60 min, but not significant between 60 min and 120 min. Organotypic spinal cord slices cultured in glucose-free medium and anaerobic incubator could mimic hypoxia-ischemia of the spinal cord perfectly; 60 min could be the best duration for OGD. This technique might be a simple and efficient method to obtain in vitro model for spinal cord hypoxic-ischemic injury in sufficient number and with high quality.

Introduction

Hypoxic-ischemic injury, as a pathophysiological change, is common in various spinal cord diseases. However, the pathophysiology of the lesion is not clear. To date, there is a paucity of ideal experimental models for the investigation of this problem. The two classical protocols have been commonly used for establishing hypoxic-ischemic spinal cord injury. One is in vivo model (Dionne and Tyler, 2013, Humpel, 2015, Sypecka et al., 2015), which is difficult to establish and usually requires considerable amount of experimental animals and time. The complexity of in vivo system also makes it difficult to identify individual environmental factors (Humpel, 2015, Kim et al., 2010). The other is in vitro model (Cifra et al., 2012, Kim et al., 2010, Sypecka et al., 2015), which involves cell culture and oxygen glucose deprivation (OGD) in organotypic spinal cord slices. The complexity of cellular and molecular composition usually can hardly be mimicked by dissociated cell culture system, but the OGD model in organotypic spinal cord slices can preserve the basic tissue cytoarchitecture as well as closely mimic complex tissue microenvironment (Cifra et al., 2012, Dionne and Tyler, 2013, Humpel, 2015, Kim et al., 2010, Sypecka et al., 2015). Currently, it has become an esstential method for research of hypoxia and ischemia in spinal cord.

OGD model in organotypic brain slices used in research of neuroscience has been widely reported (Calabresi et al., 1999, Chauhan et al., 2013, Detert et al., 2013, Girard et al., 2013, Humpel, 2015, Llorente et al., 2015, McCarran and Goldberg, 2007, Riew et al., 2015, Saleem et al., 2009, Wise-Faberowski and Osorio-Lujan, 2013, Yin et al., 2015). Comparatively, only a limited number of studies have concerned the model in organotypic spinal cord slices (Cifra et al., 2012, Galante et al., 2000, Kim et al., 2010, Krassioukov et al., 2002, Mazzone and Nistri, 2011, Sypecka et al., 2013, Sypecka et al., 2015, Takuma et al., 2002). In addition to the rarity of relative literatures (Goncharenko et al., 2014), the focus of these studies is rather acute spinal cord slices, in which the slices are OGD a short time after they are sectioned, than long-term cultured slices. Furthermore, details of the OGD model production have not been discussed sufficiently and the duration of OGD remains unclear. The changes of biochemical parameters are uncertain, and the degree of spinal cord hypoxic-ischemic injury induced by different OGD durations has not been evaluated. To better understand the above issues, we conducted the present study to establish an OGD model in organotypic spinal cord slices. Different durations of OGD were applied and the degrees of hypoxic-ischemic injury in different OGD duration were evaluated. Our goal was to identify the best possible parameters for establishing an OGD model that could closely mimic spinal cord hypoxic-ischemic injury and had stable quality, high production and high reproducibility.

Section snippets

Ethical statement

Protocols for animal care and experimental management were approved by the Xi'an Jiaotong University Animal Experimentation Committee. Ethical approval for the study was obtained from the Ethics Committee of the Second Affiliated Hospital, Xi'an Jiaotong University. Principles of laboratory animal care were followed, and all experimental procedures were conducted according to guidelines established by the National Institutes of Health. All efforts were made to minimize the number of animals

Results

The organotypic spinal cord slices had clear structure and were semi-transparent with bright and good refraction until the 14th day of culture. The viability of the slices was excellent as assessed by the trypan blue exclusion method since only a small number of cells appeared blue. The slices became dim with decreased refraction in the OGD process. The longer the OGD was, the more obvious the changes were. The structure and refraction disappeared in the OGD 120 min group, similar as in positive

Discussion

This study presents a simple and efficient method to establish OGD model in organotypic spinal cord slices with high production and stability. We show for the first time that OGD model in long-term cultured organotypic spinal cord slices can closely mimic hypoxic-ischemic injury of spinal cord. OGD for 60 min simulates hypoxic-ischemic injury excellently, proven by morphological changes, PI uptake test, LDH quantification and western blotting. This suggests that 60 min is relatively the best

Conflict of interest

The authors declare that there are no conflicts of interest.

Acknowledgments

This study was supported by National Natural Science Foundation of China (81471247 and 81271340).

References (24)

  • A. Cifra et al.

    Postnatal developmental profile of neurons and glia in motor nuclei of the brainstem and spinal cord, and its comparison with organotypic slice cultures

    Dev. Neurobiol.

    (2012)
  • J.A. Detert et al.

    Pretreatment with apoaequorin protects hippocampal CA1 neurons from oxygen-glucose deprivation

    PLoS One

    (2013)
  • Cited by (0)

    View full text