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Investigating neurodegeneration after birth injury – insights from touchscreen experiments

Sein Cho

Johns Hopkins University

Neonatal Hypoxia-Ischemia Encephalopathy (nHIE) is a devastating birth complication that goes on to affect infants in their long-term development as well as cognition. The lab uses mice models to further study the correlation between nHIE and cognitive development. I was mainly working with behaivoral testing of mice, and especially with the touchscreen platform. To induce hypoxic-ischemic (HI) injury, the Vannucci model was employed, involving unilateral right carotid ligation. HI mice experienced 45 minutes of hypoxia with a fraction of inspired oxygen (FiO2) of 0.08, along with ligation and subsequent exposure to an 8% FiO2 hypoxic chamber, simulating HI injury akin to human birth conditions. Sham mice, on the other hand, did not undergo HI surgery but were anesthetized and exposed to an FiO2 of 0.21 in a comparable environment to the HI mice. Reflex testing was conducted between pages 11 and 21. Light dark box tests were administered on page 30, followed by open field and Y-maze tests on page 60. Subsequently, mice underwent food restriction and were tested using the touchscreen platform to evaluate visual discrimination and reversal learning.


Hypoxia-Ischemia Encephalopathy (HIE) is perinatal loss of O2 and blood flow to brain. It typically leads to impairment in learning, memory development, including conditions such as cerebral palsy, epilepsy, secondary microcephaly, learning disabilities, etc. It is also known for producing delays in learning, memory development. The lab investigates neurodegeneration after birth injury, and especially the hippocampus’s role in learning, memory, various cognitive functions after neonatal HIE. Continued studies and experiments are undergoing to explore the effect of nHIE in brain and cognitive development.



Induced HI

To stimulate HI, the Vannucci model was used to undergo unilateral right carotid ligation. The HI mice underwent 45 min hypoxia FiO2= 0.08 (1,2), with a ligation followed by a time in 8% FiO2 hypoxic chamber to stimulate HI injury during human birth. The sham mice did not receive surgery for HI but were anesthetized and exposed to FiO2= 0.21 in the same environment as HI mice (Nguyen et al., 2015).


Behavioral Testing

MedAssociates touchscreen boxes equipped with K-Limbic software were used to evaluate learning and memory capacity. The testing process included 4 training stages (acclimation, bar press, touch training, and punishment) and 2 assessment stages: visual discrimination and reversal learning.



Puppies were placed in one arm of a Y-maze for 5 minutes and given freedom to explore all three arms. A software analyzed their movements and determined the percentage of spontaneous arm changes compared to total arm entries. Typical behavior involves alternating through all three arms before revisiting a previously explored one. One week following phase 1, phase 2 was conducted to evaluate memory over a longer period. Mice were allowed 5 minutes to explore two arms of the apparatus, while the third was inaccessible. After at least 20 minutes, mice were reintroduced to the apparatus for another 5 minutes, now with all three arms accessible. Mice typically favor the new arm since it was previously blocked off, and phase two examines whether they recall this prior obstruction. Between postnatal days 55 and 60, open field testing was conducted to evaluate anxiety-related behaviors and locomotion in mice. The mice were placed in a box with the center marked with tape, and the duration spent on the edges outside the taped area was recorded. Increased time spent in the corners and on the edges suggests higher levels of anxiety. Additionally, the total distance traveled was measured as an indicator of locomotion.

Figure 1. A sample model of touchscreen used for behavioral testing  (EdSpace)

Timeline of experimental design of the study (adapted from Maxwell et al.). Mice randomized to the HIE group underwent the carotid artery ligation and hypoxia chamber exposure at postnatal day 10 (P10). Following recovery, the touchscreen cognitive testing started with pretraining, followed by discrimination and reversal tasks in both sham and HIE groups. During pretraining, mice became familiar with the chamber and the food reward system. In discrimination, one symbol was the correct answer, which would result in a food reward if chosen. During reversal, the symbol that was previously correct became the incorrect response.

Touchscreen assessment

Once mice reach a minimum weight of 18g, they commence food restriction. The aim is for them to achieve 85% of their weight prior to restriction, ensuring their health while creating a motivation for pellet rewards in the touchscreen platform. Following one week of food restriction, mice undergo training in the touchscreen apparatus before actual testing begins. This training teaches them where to find the reward and the actions required to obtain it. Once they've mastered the sequence for obtaining the reward (such as pressing a lever or touching the correct picture with their nose), they proceed to the initial phase of evaluation: visual discrimination.


In the visual discrimination task, mice are presented with two unfamiliar stimuli. One stimulus leads to a food reward accompanied by a tone, while the other stimulus leads to a 10-second timeout without any reward. To pass the discrimination test, mice must make at least 85% of their responses towards the stimulus associated with the reward for two consecutive days. This test assesses the mouse's capacity to learn which image leads to the reward and which does not. Once the test is successfully completed, mice proceed to reversal learning. Such tests assess memory, cognition, and spatial recognition


On p180 the mice models were perfused for fresh tissue collection and further analysis.

The table below summarizes the stages.



Test Description

Passing Criteria 



Mouse is familiarized with the touchscreen boxes and the testing environment. 10 food pellets are placed in the feeding chamber.

Mouse must eat all 10 pellets loaded into the feeding chamber in the 30 minutes.



Bar Press

Mouse must press down on the bar press to receive a food pellet reward.

Mouse must complete 30 trials in 30 minutes.


Touch Training

Mouse must first press the bar and then one of two touchscreens to receive a reward.

Mouse must complete 30 trials in thirty minutes. 



Mouse must touch the bar and then the appropriate screen while the “fan" image is presented (correct trial) to receive a food pellet reward. If the mouse instead touches the blank screen (incorrect trial), the mouse is punished by exposure to a bright light and high-pitched noise for five seconds. 

Mouse must receive a score of at least 23 correct trials out of 30. 


Visual Discrimination

Mouse must touch the bar and then the fan image (correct trial) when presented alongside the marble image on the touchscreen to receive a food pellet reward. If the mouse touches the marble image (incorrect trial), the mouse is punished with exposure to a bright light and high-pitched noise for five seconds.

Mouse must receive an average score of 25.5/30 across two consecutive days of testing with a minimum score of 25/30 in that same period.


Reversal Learning

Mouse must touch the bar and then the marble image (correct trial) when presented alongside the fan image on the touchscreen to receive a food pellet reward. If the mouse touches the fan image (incorrect trial), the mouse is punished with exposure to a bright light and high-pitched noise for five seconds.

Mouse must receive an average score of 25.5/30 across two consecutive days of testing with a minimum score of 25/30 in that same period.


Results and Discussion


The animals are still in the process of undergoing behavioral testing, and further analyses is needed to provide a definitive conclusion. Analysis of results from behavioral testing based on genotype & treatment status (HI vs Sham) could potentially show a possible correlation btw cognitive degeneration and neonatal HI. Such data could get us closer to answering questions such as how nHIE relates to neurodegeneration in adults as well as the long-term effects of nHIE. Overall, the project will allow us to explore the correlation between neonatal brain injury and adult neurodegenerative disease.




Nguyen, T. V., Crumpacker, R. H., Calderon, K. E., Garcia, F. G., Zbesko, J. C., Frye, J. B., Gonzalez, S., Becktel, D. A., Yang, T., Tavera-Garcia, M. A., Morrison, H. W., Schnellmann, R. G., Longo, F. M., & Doyle, K. P. (2022). Post-Stroke Administration of the p75 Neurotrophin Receptor Modulator, LM11A-31, Attenuates Chronic Changes in Brain Metabolism, Increases Neurotransmitter Levels, and Improves Recovery. The Journal of pharmacology and experimental therapeutics, 380(2), 126–141.


Maxwell JR, Zimmerman AJ, Pavlik N, Newville JC, Carlin K, Robinson S, Brigman JL, Northington FJ and Jantzie LL (2020) Neonatal Hypoxic-Ischemic Encephalopathy Yields Permanent Deficits in Learning Acquisition: A Preclinical Touchscreen Assessment. Front. Pediatr. 8:289. doi: 10.3389/fped.2020.00289


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