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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

Schematic diagram and dimensions of the custom-designed stereotaxic frame used in the 2P-IBI model. (A) Section view. Cross section view shows the mounting groove engaged with an ear bar. (B) Top view. Position of the heavy base plate, U frame, 2 clamps, 6 CSK screws as well as the 2 M4 screws are shown. Position of the heating pad is depicted. (C) Side view. Thickness of the base plate and U frame are shown. Red circle shows a clamp gripping an ear bar. Red box shows a magnified view of the clamp and the inclined wedge gripping the ear bar. Note the notch of the ear bar slips into a groove in the underbelly of the clamp. (D) 3-dimensional view of the frame. (E–H) Angle views of the clamp. (E) Side view with grooves in the underbelly, (F) 3-dimensional view with a hole for fitting the M4 screw, (G) Top view, (H) Ridges carved on the inclined wedge to grip the ear bar.
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Figure 1: Schematic diagram and dimensions of the custom-designed stereotaxic frame used in the 2P-IBI model. (A) Section view. Cross section view shows the mounting groove engaged with an ear bar. (B) Top view. Position of the heavy base plate, U frame, 2 clamps, 6 CSK screws as well as the 2 M4 screws are shown. Position of the heating pad is depicted. (C) Side view. Thickness of the base plate and U frame are shown. Red circle shows a clamp gripping an ear bar. Red box shows a magnified view of the clamp and the inclined wedge gripping the ear bar. Note the notch of the ear bar slips into a groove in the underbelly of the clamp. (D) 3-dimensional view of the frame. (E–H) Angle views of the clamp. (E) Side view with grooves in the underbelly, (F) 3-dimensional view with a hole for fitting the M4 screw, (G) Top view, (H) Ridges carved on the inclined wedge to grip the ear bar.

Mentions: The custom-designed stereotaxic frame used in this protocol consists of a heavy aluminium base plate, “U” frame and 2 ear bar clamps and is most suited for 6–8-week old mice (Figures 1, 2). The bulky base helps to provide stability for operator use. The “U” frame is bolted to the base plate via 6 CSK screws fitted from beneath. The clamps are then fitted in place using M4 CSK screws. Ear bars with 18° or 45° points may be used. The 18° ear bars provide good head restraint but can cause internal ear hematoma and acute inflammation (Prestwich et al., 2008). We use the non-rupture wide angle 45° points that provide adequate head restraint and carry minimal risk of injury or inflammation. Mount ear bars into the mounting groove. Once assembled, the contraption can be stored in this configuration. A tooth bar and nose clamp did not provide additional stability for head restraint in our experiments.


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)

Schematic diagram and dimensions of the custom-designed stereotaxic frame used in the 2P-IBI model. (A) Section view. Cross section view shows the mounting groove engaged with an ear bar. (B) Top view. Position of the heavy base plate, U frame, 2 clamps, 6 CSK screws as well as the 2 M4 screws are shown. Position of the heating pad is depicted. (C) Side view. Thickness of the base plate and U frame are shown. Red circle shows a clamp gripping an ear bar. Red box shows a magnified view of the clamp and the inclined wedge gripping the ear bar. Note the notch of the ear bar slips into a groove in the underbelly of the clamp. (D) 3-dimensional view of the frame. (E–H) Angle views of the clamp. (E) Side view with grooves in the underbelly, (F) 3-dimensional view with a hole for fitting the M4 screw, (G) Top view, (H) Ridges carved on the inclined wedge to grip the ear bar.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Schematic diagram and dimensions of the custom-designed stereotaxic frame used in the 2P-IBI model. (A) Section view. Cross section view shows the mounting groove engaged with an ear bar. (B) Top view. Position of the heavy base plate, U frame, 2 clamps, 6 CSK screws as well as the 2 M4 screws are shown. Position of the heating pad is depicted. (C) Side view. Thickness of the base plate and U frame are shown. Red circle shows a clamp gripping an ear bar. Red box shows a magnified view of the clamp and the inclined wedge gripping the ear bar. Note the notch of the ear bar slips into a groove in the underbelly of the clamp. (D) 3-dimensional view of the frame. (E–H) Angle views of the clamp. (E) Side view with grooves in the underbelly, (F) 3-dimensional view with a hole for fitting the M4 screw, (G) Top view, (H) Ridges carved on the inclined wedge to grip the ear bar.
Mentions: The custom-designed stereotaxic frame used in this protocol consists of a heavy aluminium base plate, “U” frame and 2 ear bar clamps and is most suited for 6–8-week old mice (Figures 1, 2). The bulky base helps to provide stability for operator use. The “U” frame is bolted to the base plate via 6 CSK screws fitted from beneath. The clamps are then fitted in place using M4 CSK screws. Ear bars with 18° or 45° points may be used. The 18° ear bars provide good head restraint but can cause internal ear hematoma and acute inflammation (Prestwich et al., 2008). We use the non-rupture wide angle 45° points that provide adequate head restraint and carry minimal risk of injury or inflammation. Mount ear bars into the mounting groove. Once assembled, the contraption can be stored in this configuration. A tooth bar and nose clamp did not provide additional stability for head restraint in our experiments.

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