Elsevier

Brain Research Protocols

Volume 13, Issue 3, August 2004, Pages 189-196
Brain Research Protocols

Protocols
Krypton laser-induced photothrombotic distal middle cerebral artery occlusion without craniectomy in mice

https://doi.org/10.1016/j.brainresprot.2004.06.001Get rights and content

Abstract

Recent advances in genetical engineering of the mouse have highlighted the importance of reproducible and less invasive models of cerebral ischemia in mice. In this paper, we developed minimally invasive and reproducible model of distal middle cerebral artery (MCA) occlusion in mice using krypton (Kr) laser-induced photothrombosis.

C57BL/6 or BALB mice (n=8 each) were anesthetized with halothane. The skin was cut, the temporal muscle was retracted, and the right distal MCA was observed through the skull. A Kr laser beam of wavelength 568 nm was focused onto the MCA over the intact skull. Upon laser irradiation, intravenous administration of a rose bengal solution was begun. After 4 min of irradiation, the laser beam was refocused on the MCA just proximal to the first spot, and another 4-min irradiation was performed. Then, the right common carotid artery (CCA) was ligated. Three days later, the brain was removed, and infarct volume was determined.

Infarction confined almost solely to the cortical area was produced in each mouse. Mean infarct volume in C57BL/6 mice was 25.2±13.7 mm3. The BALB mice group showed significantly larger and more reproducible infarction (44.1±5.2 mm3; the coefficient of variation was 12%) than did C57BL/6 mice (P<0.005).

Our photothrombosis model of stroke in mice can be performed without craniectomy, and its reproducibility is satisfactory when using BALB mice.

Section snippets

Type of research

Focal ischemia model in mice [8], [11].

Thrombotic occlusion induced by krypton (Kr) laser and rose bengal solution [4], [23], [26].

Time required

The time required for the experiment greatly depends on the skills and practice of the researcher:

  • Stroke surgery: 45 min

  • Harvesting and sectioning the brain: 15 min

  • Staining slices of the brain: 30 min

  • Postfixing the slices: 3 days

  • Infarct volume measurement: 15 min including image capturing

Materials

All procedures were done in accordance with the Animal Care Guidelines at Kyushu University and the law (No.105) and notification (No.6) of the Japanese government.

Adult male BALB/cA Jcl (BALB) mice or C57BL/6 Jcl (C57BL/6; 8–9 weeks old, body weight 25–30 g) purchased from Nippon CLEA (Shizuoka, Japan) were used in this study.

Stroke surgery

Focal cerebral infarction was produced by permanent occlusion of the right middle cerebral artery (MCA) by a laser-induced photochemical reaction (see review by Watson [22]) modified from the rat model developed in our laboratory [4], [26]. This method enables us to make a pinpoint occlusion of a vessel leaving the dura and the skull intact and without producing massive heat. The mice were anesthetized by inhalation of 2% halothane in 70% N2O and 30% O2, and anesthesia was then maintained by

Results

In the present study, we applied Kr laser-induced photothrombosis to mice and developed a model of distal MCA occlusion while leaving the skull and dura intact. To validate this method, we produced focal ischemia in the widely used C57BL/6 Jcl and BALB/cA Jcl mouse strains with laser photothrombosis; and then, we determined whether the differences of infarct size were compatible with the results in other reports using direct cauterization of MCA.

By Kr laser-induced photothrombosis, the distal

Discussion

Recent advances in genetical engineering of the mouse and availability of a draft sequence of the mouse genome [17], as well as the availability of “knockout” or “transgenic” mice, have enabled investigators to examine effects of a given gene and its translated product in vivo. In the field of cerebral ischemia, Kinouchi et al. [11] employed the SOD1 transgenic mouse and induced focal cerebral ischemia by electric cauterization of the distal middle cerebral artery (MCA). Huang et al. [8]

Essential references

[1], [4], [22], [26], [27] are recommended for additional reading.

Quick procedure

  • 1.

    Anesthetize mouse by halothane inhalation

  • 2.

    Put suture around the right CCA and insert a catheter into the right jugular vein

  • 3.

    Retract the right temporal muscle

  • 4.

    Identify the distal part of the MCA

  • 5.

    Focus a Kr laser beam onto the MCA through the skull

  • 6.

    Inject a rose bengal solution intravenously and irradiate the MCA

  • 7.

    Tie up the suture around the right CCA

  • 8.

    Remove the catheter and close the wound

  • 9.

    Remove the brain 3 days later

  • 10.

    Slice the brain according to brain matrix

  • 11.

    Stain the slices by TTC staining

  • 12.

    Capture

Acknowledgements

The authors have declared that no conflict of interest exists. The authors are grateful to Professor Brant D. Watson for his valuable comment on this paper and for critical reading of the manuscript. We are also thankful to Drs. Junichi Takada, Yasuhiro Kumai for their technical assistance.

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