To begin this procedure, P six mice are anesthetized with indirect cooling on ice to the point of unresponsiveness to noxious stimulation. Under a dissecting microscope, the right common carotid artery is approached. A surgical hook is used to isolate the common carotid artery from the nearby vagus nerve and sympathetic ganglia.
The common carotid artery is then cauterized. Hi, I am hin from the lefty of bin in the Department of Cell Biology and Human Anatomy at University of California Davis. Hi, I'm Jennifer Plain.
I'm also from the Laboratory of one Ang. Today we will show you the procedure for creating a mouse model of Perticular lulac PVL. The predominant form of brain injury in premature infants is characterized by paraventricular.
We meta lesions. We use P six mice to create a PVL like injury by unilateral car ligation and hypoxia with and result injection of LPS. We use this procedure in our laboratory to study the pathology of PVL.
Establishing mouse models of PVL will greatly facilitate the study of disease pathogenesis using available transgenic mouse strains. These models will facilitate the conduction of drug trials in a relatively high throughput manner to discover candidate therapeutic agents as well as testing stem cell transplantation and immunodeficiency mouse strains. So let's get started.
Prior to the surgery for creating neonatal brain injury, perform a foot pinch to confirm that the animal is completely anesthetized. Clean the skin around the neck of the anesthetized P six mouse with alcohol. Then make a midline ventral incision in the anterior neck.
Under a dissecting microscope, gently retract the omohyoid and sternocleidomastoid muscles to access the right common carotid artery. Remove the fascia around the carotid sheath. Then use a hook to isolate the common carotid artery from the nearby vagus nerve and sympathetic ganglia.
Now cauterize the common carotid artery following cauterization. Close the skin incision with tissue glue. Use a heating pad to keep the animal warm until it is fully awake, and then return it to the dam.
One hour after the carotid ligation, place the mouse in a sealed chamber infused with nitrogen until a level of 6%oxygen is reached. Expose the animal to 35 minutes of hypoxia after hypoxia exposure, allow the mouse to recover on heating pad for 30 minutes and then return it to the dam for creation of brain injury with combined hypoxia, ischemia and infection.Inflammation. The animal is injected intitally with 0.015 milliliters of lipopolysaccharide and then return to the dam.
Here are some representative results from mouse models of per ventricular leukomalacia or PVL. First, a neuropathological examination of coronal sections stained with hemat, toin, and asin through the entire forebrain reveals focal necrosis in the central white matter of the cerebral hemisphere ipsilateral to the carotid ligation in the focal necrotic white matter. There is marked increase in cellularity consisting of macrophages and reactive astrocytes as this characteristic of focal human PVL in the contralateral central white matter.
The oligo den leal nuclei are arranged in fascicular like bundles. This architecture is completely disrupted by the focal necrosis on the ipsilateral side. In brains with ipsilateral cerebral white matter injury, ipsilateral necrosis in the hippocampus and focal micro infarcts in the thalamus are also observed.
Next immuno cyto chemical detection of myelin basic protein MVP or O one antigen is used to evaluate injury. Shown here is a standard scale for scoring the white matter injury using a scale of zero to five. White matter pathology is also characterized by MVP or O one staining and myelin structure by electron microscopy MVP and O one staining show myelin loss in cerebral white matter ipsilateral to the ligation compared to contralateral white matter.
EM examination shows myelinated axons in cerebral white matter contralateral to the ligation and degenerated tissue in ipsilateral white matter. Finally, to test for correlative effects of white matter injury on functional outcomes, behavioral tests for motor function were performed as shown in this graph. Behavior scoring based on a climbing test reveals that mice exposed to hypoxia ischemia without LPS have significantly lower scores compared to P 14 normal control mice.
In addition, mice exposed to hypoxia ischemia with LPS have significantly lower scores compared to P 14. Hi mice. We've just shown you how to create a mouse model of PVL by hypoxia ischemia with or result intraperitoneal injection of A RPS.
When doing this procedure, it's important to remember to isolate the common carotid artery from other tissues before cauterization. Otherwise it will result in the injury to be too severe or cause no injury. So that's it.
Thanks for watching and good luck with your experiments.
