Chronic Window Preparations

Chronic windows developed to date include the rabbit ear chamber, dorsal skin chamber, cranial window, hamster cheek pouch, spinal window, rabbit bone chamber, mouse abdominal window, mammary window, and lung window (Table I). Each of these chronic windows has its advantages and disadvantages. For example, the rabbit ear chamber is perhaps the most optically clear. However, rabbits are expensive to purchase and maintain, and it takes four to six weeks to mature the granulation tissue before the study begins. Mice, hamsters, and rats are less expensive and require smaller quantities of reagents because of their smaller body weight. From the surgical point of view, rats and hamsters are easier to work with than mice, but the latter have many advantages. The availability of comprehensive genetic information on mice, immunodeficient and genetically engineered mice, and murine reagents such as surface markers or antibodies has made mice the most commonly used laboratory animals for modern biological research. The dorsal skin chamber in mice is the most widely used chamber preparation because the surgery is less involved than some of the other preparations and because of its longer history. The cranial window can be kept for up to a year, compared to 30 to 40 days for the dorsal skin chamber, and (along with the cheek pouch) is an immuno-privileged site. The main disadvantage of the cranial window is that, in most cases, the visualization of microvessels requires both epi-illumination and the injection of a contrast agent such as a fluorescent marker.

The Rabbit Ear Chamber

In 1924, Sandison developed the first transparent window (chamber) for implantation in the ear of a rabbit. Since then, various modifications of this model have been used for microcirculation studies. The model requires granulation tissue formation within the chamber, and during this period it is very useful for the study of the wound healing process. The chambers can be used either for normal tissue studies or for tumor implantation after the maturation of granulation tissue.

Typically, a rabbit ear chamber is surgically implanted in the 15-cm-long ears of New Zealand white rabbits (~3kg body weight). Four holes are punched in the shaved ear: three outer perforations (3.5 mm in diameter) for the chamber positioning and a central puncture (5.4 mm diameter) for the transparent window housing. A molded plate is placed on the inside of the ear and aligned with the existing holes,

Table I Examples of Window Models for Microcirculation Studies.

Model

Species

Application

Ear chamber Dorsal skin chamber

Cranial window

Spinal window Bone chamber

Abdominal window

Mammary window Lung window

Rabbits

Rabbits, rats, hamsters, mice Pigs, cats, rats, mice

Cheek pouch window Hamsters

Rats, mice Rabbits

Mice

Rats, mice Rabbits, mice

Wound healing, tumor growth Skin microcirculation, ischemia reperfusion, local cytokine application, wound healing, tumor growth, angiogenesis assay, tissue growth

Cerebral and pial microcirculation, local cytokine application, wound healing, tumor growth, angiogenesis assay, tissue growth

Cheek pouch microcirculation, wound healing, tumor growth

Spinal microcirculation

Bone microcirculation, ischemia reperfusion, wound healing, stem cell homing

Abdominal wall and pancreas microcirculation, tumor growth

Tumor growth

Lung and pleural microcirculation, tumor growth while a thin (~200 mm) cover of mica glass is positioned on the outside of the cartilage. A thin granulation tissue bed (with a thickness of ~40 mm and a diameter of ~5 mm) grows in the chamber, sandwiched between the molded plate and the mica glass. The granulation tissue develops at an average of 8 days after implantation, and reaches maturity at approximately 40 days after implantation.

For tumor implantation, the cover glass, which forms the top plate of the transparent chamber, is carefully removed. A tumor (such as VX2 carcinoma) is excised from the flank of a tumor-bearing host, minced, placed in 0.9 percent NaCl solution, and spread uniformly over the cover glass. The cover glass is replaced flush against the intact normal tissue. Angiogenic response is observed 3 to 4 days post-implant, and the tumor-bearing chamber is ready for intravital microscopy approximately 10 days after implantation.

For intravital microscopy, the animal is placed in a dorsal recumbent position in a cradle that restricts head movement while still maintaining proper circulation to the chamber. The ear containing the chamber is extended horizontally to the specimen plane of an intravital microscope. The chamber is secured to the microscope stage with an aluminum adapter.

The Dorsal Skin Chamber

In the 1940s, Algire adapted the Sandison chamber to the dorsal skin in mice and carried out pioneering studies of angiogenesis during wound healing and tumor growth. Algire's original design and its modifications for other species require granulation tissue growth similar to that in the rabbit ear chamber model. Although this type of dorsal skin chamber has better optical accessibility, the effects associated with the wound healing process may hamper the observation. Later development of new dorsal skin chamber design minimized such effects by using intact skin, as described below. This type of dorsal skin chamber has been used for immunocompetent as well as immunodeficient mice, rats, and hamsters. With the availability of genetically engineered animals, the mouse dorsal skin chamber (Figure 1) has the broadest range of applications.

For the implantation of a transparent chamber into mouse dorsal skin, the entire back of the animal is shaved and depilated and two symmetrical titanium frames (weight 3.2g) are implanted so as to sandwich the extended double layers of skin. One layer of the skin is removed in a circular area approximately 15 mm in diameter, and the remaining layer (consisting of epidermis, subcutaneous tissue, and striated muscle) is covered with a circular cover glass (11 mm in diameter) incorporated into one of the frames. Following implantation of the dorsal skin chamber, animals are allowed to recover from microsurgery for 48 hours before in vivo microscopy studies or subsequent procedures are conducted.

To implant tissue, such as a tumor, the animals are positioned in a transparent polycarbonate tube (inner diameter: 25 mm). The cover glass is carefully removed and a small piece of tissue (approximately 1 mm in diameter) or 2 ml of dense cell suspension (~2 x 105 cells) is implanted at the center of the dorsal skin chamber. A new cover glass is then placed on the chamber.

For microscopy on a dorsal skin chamber-bearing mouse, the mouse is positioned in the polycarbonate tube. A heating tape is wrapped around the plastic tube to maintain the animal's core temperature at a constant ~37°C during the observations. To obtain microcirculatory parameters, chambers are observed under an intravital microscope, which is typically equipped with a high-sensitivity camera for fluorescence imaging.

Mouse Cranial Window Fixed Animal
Figure 1 Mouse Dorsal Skin Chamber Model. An SCID mouse bearing a dorsal skin chamber (left) and a macroscopic image of the mouse dorsal skin chamber (right). The frame is made of titanium (3.2 g). The window is 11 mm across. (see color insert)
Laboratory Imaging Chamber Rodent
Figure 2 Mouse Cranial Window Model. Nude mouse bearing a cranial window (left) and a macroscopic image of the mouse cranial window (right). The window is 7 mm across. (see color insert)

The Cranial Window

The closed transparent cranial window model has been extensively used for cerebral microcirculation studies. After the recovery of central nervous system fluid and intra-cephalous pressure to normal levels, this model provides direct optical access to cerebral blood vessels under physiological condition. The cranial window is the most stable window model to date, allowing observation for as long as the entire natural life span of the animal; it is therefore very useful for studies requiring relatively long duration. Cranial windows are used for angiogenesis, vessel remodeling, and maturation studies. Furthermore, this model provides a natural microenvironment for primary and metastatic brain tumors. Various species, including mice, rats, cats, and pigs, are used for the cranial window model. We will describe the preparation of the mouse cranial window (Figure 2), but similar procedures are used for other species.

For cranial window implantation, a stereotactic apparatus holds the head of the animal. The skin is cut in a circular manner on top of the skull, and the periosteum underneath is scraped off to the temporal crests. A 6-mm circle is drawn over the skull, and a groove is made on the drawn circle using a high-speed air-turbine drill with a 0.5-mm-diameter burr tip. Drilling of the groove continues until the bone flap becomes loose. Using a malis dissector, the bone flap is separated from the dura mater underneath. A nick is made close to the sagittal sinus. Iris microscissors are passed through the nick. The dura and arachnoid membranes are cut completely from the surface of both hemispheres, avoiding any damage to the sagittal sinus. The window is sealed with a 7-mm cover glass, which is glued to the bone with histo-compatible cyanoacrylate glue.

For implantation of tumors or tissue constructs, the cover glass is removed one week after the window implantation. A small piece of tissue is inoculated at the center of the window. Alternatively, 3 to 5 ml of a thick single cell suspension (1-5 x 105 cells/ml) are implanted with a 28-gauge micro-syringe (using a needle tip angle of 55 degrees and a depth of 1.75 mm).

For intravital microscopy, an animal is anesthetized and put on a polycarbonate plate, with the head fixed by means of a metal ring upper frontal tooth holder and a bilateral plastic ear holder. A heating pad is placed under the animal to maintain its temperature at ~37°C during in vivo microscopy.

Other Window Models

A spinal window has been made in rats (C5) and mice (C1 to C7) with a laminectomy. The windows were sealed with dental acrylic resin and a 7-mm cover glass (rats) or gas-impermeable transparent membrane (mice). The dorsal surface of the cervical cord was observed for leukocyte, T-cell, and platelet interaction with spinal vessels.

The rabbit bone chamber is a hollow titanium-alloy screw with quartz lenses embedded in its core. Vessels, followed by bone tissue, grow in a 100-|mm-thick chamber between two flat quartz elements implanted in tibial bone. Intravital microscopy in the bone chamber has been used to study bone microcirculation, ischemia reperfusion injury in bone, and stem cell homing to bone marrow.

An abdominal window model for the mouse pancreas has been reported. A portion of the pancreas is gently exteriorized through a small laparotomy and kept within a space surrounded by the outer side of the abdominal wall, a titanium ring, and a circular glass cover slip (11mm in diameter) held by the titanium ring. This model permits repeated observation of microcirculation in the pancreas and of the growth and treatment of pancreatic tumors.

The mammary window in rats and mice allows chronic observation of tumors growing in the breast tissue. This model requires removal of skin, nipple, and epithelium before injection of tumors and placement of an acrylic disk. Alternately, microcirculation in intact breast tissue and/or tumors grown in the breast can be observed acutely by placing a surgically prepared tissue flap in a specially designed microscope stage or chronically by using a mammary chamber similar to the mouse dorsal skin chamber.

Lung windows in New Zealand White rabbits have been used to study pulmonary microcirculation via intravital microscopy. The smaller intact pleural window involves the removal of the skin, intercostal muscles, and endothoracic fascia to expose the costal pleura, through which the lung surface is observed. The larger type of window requires the removal of the thoracic wall, including ribs, over a 3-cm distance and the implantation of a transparent window with a hollow cylinder to apply negative pressure. The lungs are inflated by positive air pressure of 6 mmHg, and the lung surface is sucked onto the window. Hemodynamics and short-term leukocyte/platelet-endothelial interactions were studied using these rabbit lung window models.

On the other hand, the chronic thoracic window model in mice allows temporal observation of the microcirculation of tumors implanted in the lung. For the implantation, a 6-mm ventral portion of the chest wall is removed, covered with a circular glass cover slip (8 mm in diameter), and sealed with biocompatible cyanoacrylate adhesive and cement mixture. Withdrawal of the remaining air in the chest cavity allows the lung to inflate and attach to the cover slip. One week later, a tumor fragment is implanted on the pulmonary pleural surface of the window. For intravital microscopy, the animal is placed in the lateral position and the chest wall is attached to adjustable arms on the stage by cranial and caudal sutures.

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