| The model NDI uses recognizes the rocky history of previous efforts to model traumatic injury to the brain. Instead of a single endpoint measure, multiple endpoints are used in a comprehensive study design, focusing on behavioral tests with clinical relevance.
Example of a specific study design
Animals
Forty-eight adult male rats are used, plus six rats for backup in case of loss during surgical procedures. There will be four groups (n = 12) as follows:
Group 1 Lesion plus Vehicle
Group 2 Lesion plus one dose of test compound
Group 3 Sham lesion plus Vehicle
Group 4 Sham lesion plus same dose of test compound
Group identity is blinded to the investigator to reduce experimental bias. The animals are given the agent or vehicle injections IP or SC beginning immediately after controlled cortical impact injury. All animals are examined on a series of behavioral tests (tabulated below) which have been shown to be sensitive to the type of injury inflicted. Upon completion of behavioral testing, brains are harvested and cut on a cryostat for examination of lesion reconstruction and hippocampal atrophy, and for measurement of retrograde degeneration (NeuN stain for counting neurons).
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MEASURES |
OUTCOMES & ANALYSIS |
DAYS / NUMBER OF TESTS |
| 1 |
SENSORY NEGLECT
Tactile adhesive removal
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Latencies to remove the stimuli from their front paws with their mouths |
One day pre-surgery and 3, 7, 14, and 21 days post-injury |
| 2 |
MOTOR COORDINATION
accelerating rotorod
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Latency to fall off a rotorod |
One day pre-surgery following training (baseline) 3, 7, 14, and 21 days post-injury |
| 3 |
SPONTANEOUS LOCOMOTOR ACTIVITY |
Number of vertical movements
Time spent in the center of box
Time spent in the corners
Total horizontal distance traversed
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1, 3, 7, 14 and 21 days post-injury |
| 4 |
MEMORY / COGNITION
Morris water maze
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Latency to the platform
Length of the path to the platform
Exploration pattern: % total time spent in the outer vs. inner annuli
Probe trial: Time spent in the quadrant that previously contained the platform as a % of total time spent in the pool
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Daily beginning 10 days post-injury for 10 days, 4 trials each session
Probe trial given on 11th day of testing
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| 5 |
HISTOLOGY |
Measurement of hippocampal atrophy
Measurement of retrograde degeneration (Neun labeling for counting neurons)
Measurement of necrotic cavity
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Post-surgery 21 days |
Histology
In many TBI studies, neuroprotection has been evaluated almost exclusively by measuring lesion size. It is now recognized that this measure is inadequate. A test therapeutic may reduce lesion size without having any effect on neuron survival. For this reason, we quantify any neuroprotection directly by counting neurons in the lesion area. In addition, a structure connected with the damaged cortical area (the hippocampus) is measured, to see if the test compound reduces the normal TBI-induced atrophy of this structure. The importance of this measure is that hippocampus size is related to cognitive function.
Behavior
The reason multiple behaviors are measured is to ensure that any therapeutic effect is not method-specific. The reason the behaviors are measured at multiple time points it to reveal therapeutic effects in the rate of recovery.
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