Erythropoietin as a Neuroprotectant for Neonatal Brain Injury: Animal Models - Tissue-Protective Cytokines: Methods and Protocols - page 118

Table 1
Ordinal scale

Brain injury scale

Scale Acute injury (24-48 h) Chronic injury (>1 week)

0 Normal ipsilateral hemisphere Normal ipsilateral hemisphere

1 Mild edema with <25% ipsi/contra size

difference

Mild atrophy with <25% ipsi/contra
size difference

2 Moderate edema (25-50%) difference Moderate atrophy (25-50%

difference)

3 Liquefaction of 50-75% hemisphere Cystic cavitation 50-75%

hemisphere

4 Liquefaction of ³75% hemisphere Cystic cavitation ³75% hemisphere

Using a mouse brain grid, rat pup brains are coronally sliced
into 2 mm sections. Sections are incubated in 2% TTC in 0.9%
Sodium Chloride for 30 min at room temperature. TTC is removed,
and samples washed with fixative (4% Paraformaldehyde). Sections
are removed from the solution for photographing.
Further investigation into specific effects of injury or neuro-
protective treatments can be done using mRNA protein measure-
ment or immunohistochemical approaches (see Note 6).

4 Notes

1. There are many confounders that may affect the extent of
injury with this model. The age of the rodent changes which
cell lines and brain regions are more at risk from hypoxic-ischemic
injury ( 62, 63 ). Rodent pups should be sexed since gender dif-
ferences have been seen in response to hypoxic-ischemic injury
( 64 ). These animals are exquisitely sensitive to temperature
variation, and hypothermia can attenuate hypoxic-ischemic
injury; therefore, body temperature should be constant during
hypoxia-ischemia and throughout the experimental period.
The hypoxic exposure should occur within 2-3 h after ligation
of the carotid artery to allow suckling of the pups, thus avoid-
ing the attenuation of injury secondary to fasting hypoglyce-
mia ( 65 ). If larger experiments are planned, pups can be
randomly assigned to dams and will be cross fostered. This
decreases the maternal influence on experimental outcomes.
Culling the litter size to 8 pups per litter will increase unifor-
mity of pup growth and improve survival.
2. Anesthetic choice may affect outcomes, so a consistent anes-
thetic must be used across groups and when comparing groups