J Korean Neurotraumatol Soc.  2010 Dec;6(2):89-96. 10.13004/jknts.2010.6.2.89.

In Vitro Compressive Neural Injuries Produced by Centrifugal Acceleration

• Kim JH
• Moon HJ
• Kim JH
• Kwon TH
• Chung HS
• Park YK

• KMID: 2106792
• DOI: http://doi.org/10.13004/jknts.2010.6.2.89

Abstract

OBJECTIVE
We describe a technique to induce in vitro traumatic brain injury in an organotypic slice culture (OTC).
METHODS
Centrifugal forces were applied to rat hippocampal slices at strengths ranging from 300 to 7,500 g.
RESULTS
The degree of neural injury correlated with the forces applied. A morphometric analysis of hippocampal CA area revealed significant tissue expansion. A graded posttraumatic decrease in evoked field potentials was recorded from acutely prepared slices. Immediate posttraumatic cell death was identified at the base of the slice where it was attached to the porous membrane. Delayed cell death and secondary damage induced by serum deprivation, excitotoxicity, hypoxic insults, and additional impacts were investigated and observed by conventional fluorescent microscopy.
CONCLUSION
Although this static compressive model of neural injury differs in impact duration from the clinical situation, it may be useful for neurotrauma research because it is accessible, easily adjustable, and reliable.

Keyword

Centrifugal force; In vitro model; Organotypic slice; Static compression; Traumatic brain injury

MeSH Terms

Acceleration
Animals
Brain Injuries
Cell Death
Membranes
Rats
Tissue Expansion

Figure

• 

  FIGURE 1 Diagram of the apparatus used to induce centrifugal acceleration injury on acute hippocampal slices (AHSs), with a representative photomicrograph of an AHS. The floor of the Eppendorff tube is shaped by pouring melted paraffin (P) into it so that it flows parallel to the axis of rotation. The centrifugal force (arrow) acts perpendicularly to the slice.

• 

  FIGURE 2 Morphometric analyses of the CA field areas of acute hippocampal slices after centrifugal injuries. A: Examples of propidium iodide labeling (10 µg/mL) of slices before and after 1,500-7,500 g centrifugal force. B: A graph showing changes in CA field area after graded injuries. The results are presented as the mean±SE. Numbers indicate number of experiments. Asterisks indicate a significant difference between groups (p<0.05).

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  FIGURE 3 Evoked potential recordings in AHSs. A: Graph showing the amplitude of the maximally evoked orthodromic population spike in relation to the injury severity (60 s force application). B: Graph illustrating the relationship between the amplitude of maximally evoked orthodromic population spike and the duration of exposure at 2,300 g. Numbers indicate number of experiments. Asterisks indicate significant difference between groups (p<0.05). AHSs: acute hippocampal slices.

• 

  FIGURE 4 Cell death in OTCs induced by a centrifugal force of 300 g (marked by an arrow). A: Digital images of PI fluorescence in OTCs made before and for four days after injury, and at the final state of confirmed death. B: Summary graph illustrating cell death (as a percentage of the maximal level) in OTCs subjected to injury. The fluorescence emission of PI was measured over the whole hippocampal slice. Note that the peak PI uptake was developed within 24 hr of the injury. OTCs: organotypic slice culture, PI: propidium iodide.

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  FIGURE 5 Cell death in OTCs subjected to centrifugal injury. A: Representative digital images of PI fluorescence in OTCs. Widespread PI uptake was observed at 1 hr in OTCs subjected to both light (300 g) and moderate (3,000 g) centrifugal forces, at a similar intensity. Twelve hours following injury, diffuse PI uptake became indistinct and delayed degeneration began in the neuronal layers, commonly in the inner blade of the dentate gyrus and CA1. B: Summary graph illustrating cell death measured in the whole hippocampal slice. C: Summary graph showing regional differences in cell death in OTCs measured 12 hr after injury. OTCs: organotypic slice culture, PI: propidium iodide, CA: cornus ammoni, DG: dentate gyrus.

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  FIGURE 6 Representative digital images of PI fluorescence in traumatized OTCs (300 g) followed by an additional insult delivered between 12 and 24 hr later. Secondary exposure to (A) hypoxia (for 60 min) or (B) excitotoxicity (glutamate, 10 mM for 30 min) led to increased cell death in the neuronal layer both in sham-injured and injured OTCs. Secondary exposure to (C) serum deprivation (for 24 hr) or (D) additional impact (300 g) was more likely to produce diffuse patterns of cell death in traumatized OTCs. PI: propidium iodide, OTCs: organotypic slice culture.

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