Limits...
Visualizing leukocyte trafficking in the living brain with 2-photon intravital microscopy.

Pai S, Danne KJ, Qin J, Cavanagh LL, Smith A, Hickey MJ, Weninger W - Front Cell Neurosci (2013)

Bottom Line: Intracranial structures are exposed through a cranial window, and physiologic conditions are maintained during extended imaging sessions via continuous superfusion of the brain surface with artificial cerebrospinal fluid (aCSF).Experiments typically require 1-2 h of preparation, which is followed by variable periods of immune cell tracking.Our methodology converges the experience of two laboratories over the past 10 years in diseased animal models such as cerebral ischemia, lupus, cerebral malaria, and toxoplasmosis.

View Article: PubMed Central - PubMed

Affiliation: Immune Imaging Program, The Centenary Institute Newtown, NSW, Australia ; Sydney Medical School, University of Sydney Sydney, NSW, Australia.

ABSTRACT
Intravital imaging of the superficial brain tissue in mice represents a powerful tool for the dissection of the cellular and molecular cues underlying inflammatory and infectious central nervous system (CNS) diseases. We present here a step-by-step protocol that will enable a non-specialist to set up a two-photon brain-imaging model. The protocol offers a two-part approach that is specifically optimized for imaging leukocytes but can be easily adapted to answer varied CNS-related biological questions. The protocol enables simultaneous visualization of fluorescently labeled immune cells, the pial microvasculature and extracellular structures such as collagen fibers at high spatial and temporal resolution. Intracranial structures are exposed through a cranial window, and physiologic conditions are maintained during extended imaging sessions via continuous superfusion of the brain surface with artificial cerebrospinal fluid (aCSF). Experiments typically require 1-2 h of preparation, which is followed by variable periods of immune cell tracking. Our methodology converges the experience of two laboratories over the past 10 years in diseased animal models such as cerebral ischemia, lupus, cerebral malaria, and toxoplasmosis. We exemplify the utility of this protocol by tracking leukocytes in transgenic mice in the pial vessels under steady-state conditions.

No MeSH data available.


Related in: MedlinePlus

Illustration of a superfusion chamber. (A) A circular incision of 6 mm is made in the parietal bone after scalp retraction to form a cranial window (B) A magnified view of a superfusion chamber that has been glued to the cranial window. An inlet and outlet tubing is connected to the chamber to circulate warm aCSF over the cranial window.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3539661&req=5

Figure 3: Illustration of a superfusion chamber. (A) A circular incision of 6 mm is made in the parietal bone after scalp retraction to form a cranial window (B) A magnified view of a superfusion chamber that has been glued to the cranial window. An inlet and outlet tubing is connected to the chamber to circulate warm aCSF over the cranial window.

Mentions: The circulating superfusion chamber unit consists of a cap-like reservoir, PVC tubing, pump-operated circulating water bath, N2/CO2/O2 cylinder, and a beaker containing aCSF (Figure 3). The custom designed stainless steel cap-like reservoir is 2 mm high with an internal diameter of 7 mm. It has an outer rim 0.4 mm wide and 15 mm in diameter extending horizontally from the chamber. It is modified to include a concavity 8 mm in radius that will mold to the shape of the mouse skull. The chamber contains two ports, one for attachment of inlet polyethylene tubing for superfusing the brain surface with aCSF and the other for attachment of outlet polyethylene tubing that will collect aCSF after superfusion. The outlet tubing is positioned at an elevation of 10 cm above the mouse brain to maintain intracranial pressure at 5–8 mm Hg throughout the session. To begin, freshly prepared aCSF is decanted into a small beaker and placed in a peristaltic pump-operated warm circulating water bath and pre-warmed to 37°C. The operational speed of the peristaltic pump is adjusted to maintain aCSF infusion at 0.3 ml/min. The aCSF is continuously bubbled with a mixture of 12% O2, 5% CO2, and 83% N2.


Visualizing leukocyte trafficking in the living brain with 2-photon intravital microscopy.

Pai S, Danne KJ, Qin J, Cavanagh LL, Smith A, Hickey MJ, Weninger W - Front Cell Neurosci (2013)

Illustration of a superfusion chamber. (A) A circular incision of 6 mm is made in the parietal bone after scalp retraction to form a cranial window (B) A magnified view of a superfusion chamber that has been glued to the cranial window. An inlet and outlet tubing is connected to the chamber to circulate warm aCSF over the cranial window.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3539661&req=5

Figure 3: Illustration of a superfusion chamber. (A) A circular incision of 6 mm is made in the parietal bone after scalp retraction to form a cranial window (B) A magnified view of a superfusion chamber that has been glued to the cranial window. An inlet and outlet tubing is connected to the chamber to circulate warm aCSF over the cranial window.
Mentions: The circulating superfusion chamber unit consists of a cap-like reservoir, PVC tubing, pump-operated circulating water bath, N2/CO2/O2 cylinder, and a beaker containing aCSF (Figure 3). The custom designed stainless steel cap-like reservoir is 2 mm high with an internal diameter of 7 mm. It has an outer rim 0.4 mm wide and 15 mm in diameter extending horizontally from the chamber. It is modified to include a concavity 8 mm in radius that will mold to the shape of the mouse skull. The chamber contains two ports, one for attachment of inlet polyethylene tubing for superfusing the brain surface with aCSF and the other for attachment of outlet polyethylene tubing that will collect aCSF after superfusion. The outlet tubing is positioned at an elevation of 10 cm above the mouse brain to maintain intracranial pressure at 5–8 mm Hg throughout the session. To begin, freshly prepared aCSF is decanted into a small beaker and placed in a peristaltic pump-operated warm circulating water bath and pre-warmed to 37°C. The operational speed of the peristaltic pump is adjusted to maintain aCSF infusion at 0.3 ml/min. The aCSF is continuously bubbled with a mixture of 12% O2, 5% CO2, and 83% N2.

Bottom Line: Intracranial structures are exposed through a cranial window, and physiologic conditions are maintained during extended imaging sessions via continuous superfusion of the brain surface with artificial cerebrospinal fluid (aCSF).Experiments typically require 1-2 h of preparation, which is followed by variable periods of immune cell tracking.Our methodology converges the experience of two laboratories over the past 10 years in diseased animal models such as cerebral ischemia, lupus, cerebral malaria, and toxoplasmosis.

View Article: PubMed Central - PubMed

Affiliation: Immune Imaging Program, The Centenary Institute Newtown, NSW, Australia ; Sydney Medical School, University of Sydney Sydney, NSW, Australia.

ABSTRACT
Intravital imaging of the superficial brain tissue in mice represents a powerful tool for the dissection of the cellular and molecular cues underlying inflammatory and infectious central nervous system (CNS) diseases. We present here a step-by-step protocol that will enable a non-specialist to set up a two-photon brain-imaging model. The protocol offers a two-part approach that is specifically optimized for imaging leukocytes but can be easily adapted to answer varied CNS-related biological questions. The protocol enables simultaneous visualization of fluorescently labeled immune cells, the pial microvasculature and extracellular structures such as collagen fibers at high spatial and temporal resolution. Intracranial structures are exposed through a cranial window, and physiologic conditions are maintained during extended imaging sessions via continuous superfusion of the brain surface with artificial cerebrospinal fluid (aCSF). Experiments typically require 1-2 h of preparation, which is followed by variable periods of immune cell tracking. Our methodology converges the experience of two laboratories over the past 10 years in diseased animal models such as cerebral ischemia, lupus, cerebral malaria, and toxoplasmosis. We exemplify the utility of this protocol by tracking leukocytes in transgenic mice in the pial vessels under steady-state conditions.

No MeSH data available.


Related in: MedlinePlus