Early Pathological and Magnetic Resonance Detection of Cerebral Injury Using a Rat Model of Neonatal Hypoxic Ischemic Encephalopathy
Summary
The present protocol describes a rodent model of newborn hypoxic-ischemic injury for identifying early changes in cerebral tissue by gross morphology and magnetic resonance imaging. This has benefits over existing models, which can be used to study late injury but do not allow the evaluation of reproducible early changes.
Abstract
Perinatal hypoxic-ischemic encephalopathy (HIE) is an acute disease that may afflict newborns, resulting in variable long- and short-term neurodevelopmental outcomes. Early diagnosis is critical to identifying infants who may benefit from intervention; however, early diagnosis relies heavily on clinical criteria. No molecular or radiological tests have shown promise in detecting early cerebral injury. Studies have shown that magnetic resonance imaging (MRI) can show changes in both blood flow/ischemia and metabolic disruption. However, they have all been used to evaluate the secondary phase of the disease (>12 h) after the onset of the injury. Early diagnosis is critical to rapidly starting therapeutic hypothermia in eligible infants, which is currently recommended to be initiated within 6 h of birth. The rat model of hypoxic-ischemic injury was developed in 1981 and has been validated and used extensively to study changes in brain perfusion, cerebral injury markers, and morphology. However, it has primarily been used as a “late model”, evaluating injury several days after the initial ischemic insult. The model has been known to have poor sensitivity in evaluating reliable and reproducible early cerebral changes. The objective of this study was to develop a reliable model to study early gross morphological and radiological markers of HIE using pathological staining and cerebral magnetic resonance imaging/magnetic resonance spectroscopy.
Introduction
Hypoxic ischemic encephalopathy (HIE) is a devastating condition resulting from various factors in newborn infants1. Perinatal asphyxia and/or the disruption of cerebral blood flow may result in focal or global ischemic changes in the brain2. The occurrence rate is approximately 1.6 in 1,000 live births but may be as high as 12.1 in 1,000 live births in developing countries3. This condition results in high mortality (20%-50%), while 25% of those who survive are likely to suffer from a long-term neural disability such as mental retardation, epilepsy, or cerebral palsy4. The only therapeutic intervention proven effective in mild to moderate injury is therapeutic hypothermia, which must be initiated within 6 h of birth5,6,7,8,9. While this may help prevent the metabolic changes that lead to secondary injury, there may also be potential for side effects such as hypotension, thrombocytopenia, prolonged coagulation time, intracranial hemorrhage, dysrhythmias, fat necrosis, and serum electrolyte imbalance4,5. Early diagnosis of HIE in babies is often difficult as the criteria are subjective and rely heavily on physical exam findings, which evolve over time. Magnetic resonance imaging may show changes reflective of injury several days to weeks after injury. However, morphologic changes in T1/T2 MRI can be normal in up to two-thirds of moderate encephalopathy, the category of infants most likely to benefit from therapeutic hypothermia10. As per recent reports, magnetic resonance spectroscopy (MRS) may show early changes correlating with neonatal HIE11. However, no standardization or validation has been performed to date.
Many investigators rely on animal models to evaluate potential diagnostic or therapeutic interventions for cerebrovascular injury. The most frequently used method to create an infarct is ligating rodents' middle cerebral artery12,13. While often used to study adult ischemic stroke, this is technically challenging in neonatal rodents due to the small size and the fragility of the pups at the age equivalent to human newborn disease. Furthermore, it does not represent the global cerebral ischemic changes likely to be seen in HIE. The Rice-Vanucci Model14 of unilateral carotid artery ligation in rats has been used since the 1980s as a cost-effective rodent model to study hypoxic-ischemic brain injury. However, there is large variability in early cerebrovascular changes and high mortality in earlier experiments. Most studies report the cerebral injury in long-term changes (i.e., after 24 h of injury), which are more consistent. This study aimed to develop an approach to evaluate early (within 6 h) molecular and radiological changes in a rat model of HIE. The protocol was designed to ensure ischemia at an early (term newborn equivalent) age and to increase the survival of the pups, especially during exposure to hypoxia. MRI/MRS were used to evaluate radiological evidence of altered flow, cerebral tissue changes, and metabolic changes within 6 h of injury. Gross morphological evaluation of the infarct areas was also performed. Further validation of the reproducibility was conducted by repeating the experiments in multiple litters.
Protocol
Representative Results
Discussion
A research protocol in newborn rat pups was successfully designed to visualize and analyze early markers of cerebral injury in HIE. To date, there is a lack of objective assessment tools to detect early cerebral injury in the newborn population. After HI injury, there is a phase (1-6 h) in which the impairment of cerebral oxidative metabolism has the potential to partially recover before the failure of mitochondrial function19, which is irreversible. This latent phase is the therapeutic window for…
Disclosures
The authors have nothing to disclose.
Acknowledgements
We thank the veterinary staff of the Oklahoma Medical Research Foundation for their expertise and assistance in modifying the animal care protocols.
Materials
| 0.9% Normal saline | Fisher Scientific | Z1376 | |
| 2,3,5-triphenyltetrazolium chloride (TTC) | Millipore Sigma | T8877 | |
| Abdominal pneumatic pillow | SA Instruments, Inc., Stony Brook, NY | ||
| Absorbent Underpads with Waterproof Moisture Barrier, 58.4 x 91.4 cm, 680 mL | Fisher Scientific | 501060566 | |
| BD 30 G Needle and syringe | Fisher Scientific | Catalog No.14-826-10 | |
| Biospec 7.0 Tesla/30 cm horizontal-bore magnet small animal imaging system | Bruker Biospin, Ettlingen, Germany | ||
| Buprenorphine | Provided by veterenary medicine | ||
| Compact Thermometer with Probe | Fisher Scientific | S01549 | |
| Gas mixture 92% nitrogen 8% oxygen | Airgas | ||
| Head surface coil | Bruker BioSpin MRI Gmbh, Ettlingen, Germany | ||
| Isoflurane gas | Provided by veterenary medicine | ||
| Isotemp Immersion Circulator 2100 | Fisher Scientific | Discontinued | Immersed in water bath chamber with continous flowing water via tubing |
| Lead Ring Flask Weights | VWR | 29700-060 | Water bath weights to ensure rodent chamber stays submerged in water bath |
| Mathematica Software | Wolfram Research, Champaign, IL, USA | version 6.0 | |
| Pedialyte Electrolyte Solution, Hydration Drink, 1 Liter, Unflavored | Pedialyte | Obtained from CVS | |
| Phosphate-buffered saline (DPBS, 1X), Dulbecco's formula | Millipore Sigma | J67670.AP | |
| Plastic clear bucket | We used an old rodent housing cage- this is a good alternative: Cambro 182615CW135 Camwear Food Storage Box, 18" X 26" X 15", Model #:182615CW135 | ||
| Plexiglass Rodent Restraint Chamber | Pedialyte/CVS | Vetinary medicine provided a small chamber used to restrain rodents. Approximately 6x4x4 inches | |
| Pregnant Sprague Dawley rats at E14 | Charles River | Strain Code 400 | |
| Purdue Products Betadine Swabsticks | Fisher Scientific | 19-061617 | |
| Quadrature volume coil (72-mm inner diameter) | Bruker BioSpin MRI Gmbh, Ettlingen, Germany | ||
| Stoelting Silk Suture | Fisher Scientific | Catalog No.10-000-656 | |
| Vicryl 5-0 suture | Fisher Scientific | NC1985424 |
References
- Douglas-Escobar, M., Weiss, M. D. Hypoxic-ischemic encephalopathy: A review for the clinician. JAMA Pediatrics. 169 (4), 397-403 (2015).
- Bano, S., Chaudhary, V., Garga, U. C. Neonatal hypoxic-ischemic encephalopathy: A radiological review. Journal of Pediatric Neurosciences. 12 (1), 1-6 (2017).
- Lee, A. C., et al. Intrapartum-related neonatal encephalopathy incidence and impairment at regional and global levels for 2010 with trends from. Pediatric Research. 74, 50-72 (2013).
- American College of Obstetricians and Gynecologists Task Force on Neonatal Encephalopathy. Executive summary: Neonatal encephalopathy and neurologic outcome, second edition. Report of the American College of Obstetricians and Gynecologists Task Force on Neonatal Encephalopathy. Obstetrics & Gynecology. 123 (4), 896-901 (2014).
- Jacobs, S. E., et al. Whole-body hypothermia for term and near-term newborns with hypoxic-ischemic encephalopathy: A randomized controlled trial. Archives of Pediatrics and Adolescent. 165 (8), 692-700 (2011).
- Drury, P. P., Gunn, E. R., Bennet, L., Gunn, A. J. Mechanisms of hypothermic neuroprotection. Clinics in Perinatology. 41 (1), 161-175 (2014).
- Higgins, R. D., et al. Hypothermia and other treatment options for neonatal encephalopathy: An executive summary of the Eunice Kennedy Shriver NICHD workshop. The Journal of Pediatrics. 159 (5), 851-858 (2011).
- Patel, S. D., et al. Therapeutic hypothermia and hypoxia-ischemia in the term-equivalent neonatal rat: characterization of a translational preclinical model. Pediatric Research. 78 (3), 264-271 (2015).
- Park, W. S., et al. Hypothermia augments neuroprotective activity of mesenchymal stem cells for neonatal hypoxic-ischemic encephalopathy. PLoS One. 10 (3), 0120893 (2015).
- Agut, T., et al. Early identification of brain injury in infants with hypoxic ischemic encephalopathy at high risk for severe impairments: Accuracy of MRI performed in the first days of life. BMC Pediatrics. 14, 177 (2014).
- Guo, L., et al. Early identification of hypoxic-ischemic encephalopathy by combination of magnetic resonance (MR) imaging and proton MR spectroscopy. Experimental and Therapeutic Medicine. 12 (5), 2835-2842 (2016).
- Ashwal, S., Cole, D. J., Osborne, S., Osborne, T. N., Pearce, W. J. A new model of neonatal stroke: reversible middle cerebral artery occlusion in the rat pup. Pediatric neurology. 12 (3), 191-196 (1995).
- Larpthaveesarp, A., Gonzalez, F. F. Transient middle cerebral artery occlusion model of neonatal stroke in P10 rats. Journal of Visualized Experiments. (122), e54830 (2017).
- Rice, J. E., Vannucci, R. C., Brierley, J. B. The influence of immaturity on hypoxic-ischemic brain damage in the rat. Annals of Neurology. 9 (2), 131-141 (1981).
- Bozza, F. A., et al. Sepsis-associated encephalopathy: A magnetic resonance imaging and spectroscopy study. Journal of Cerebral Blood Flow & Metabolism. 30 (2), 440-448 (2010).
- Garteiser, P., et al. Multiparametric assessment of the anti-glioma properties of OKN007 by magnetic resonance imaging. Journal of Magnetic Resonance Imaging. 31 (4), 796-806 (2010).
- Towner, R. A., et al. Anti-inflammatory agent, OKN-007, reverses long-term neuroinflammatory responses in a rat encephalopathy model as assessed by multi-parametric MRI: implications for aging-associated neuroinflammation. Geroscience. 41 (4), 483-494 (2019).
- Adeghe, A. J., Cohen, J. A better method for terminal bleeding of mice. Lab Animal. 20 (1), 70-72 (1986).
- Rodriguez, M., Valez, V., Cimarra, C., Blasina, F., Radi, R. Hypoxic-ischemic encephalopathy and mitochondrial dysfunction: Facts, unknowns, and challenges. Antioxiddants & Redox Signaling. 33 (4), 247-262 (2020).
- Vannucci, R. C., Towfighi, J. Experimental models of hypothermic circulatory arrest. Seminars in Pediatric Neurology. 6 (1), 48-54 (1999).
- Sarkar, S., Barks, J. D. Systemic complications and hypothermia. Seminars in Fetal and Neonatal Medicine. 15 (5), 270-275 (2010).
- Chiamulera, C., Terron, A., Reggiani, A., Cristofori, P. Qualitative and quantitative analysis of the progressive cerebral damage after middle cerebral artery occlusion in mice. Brain Research. 606 (2), 251-258 (1993).
- Hatfield, R. H., Mendelow, A. D., Perry, R. H., Alvarez, L. M., Modha, P. Triphenyltetrazolium chloride (TTC) as a marker for ischaemic changes in rat brain following permanent middle cerebral artery occlusion. Neuropathology and Applied Neurobiology. 17 (1), 61-67 (1991).
- Goldlust, E. J., Paczynski, R. P., He, Y. Y., Hsu, C. Y., Goldberg, M. P. Automated measurement of infarct size with scanned images of triphenyltetrazolium chloride-stained rat brains. Stroke. 27 (9), 1657-1662 (1996).
- Robertson, N. J., Thayyil, S., Cady, E. B., Raivich, G. Magnetic resonance spectroscopy biomarkers in term perinatal asphyxial encephalopathy: From neuropathological correlates to future clinical applications. Current Pediatric Reviews. 10 (1), 37-47 (2014).
- Gano, D., et al. Evolution of pattern of injury and quantitative MRI on days 1 and 3 in term newborns with hypoxic-ischemic encephalopathy. Pediatric Research. 74 (1), 82-87 (2013).
- da Silva, L. F., Hoefel Filho, J. R., Anes, M., Nunes, M. L. Prognostic value of 1H-MRS in neonatal encephalopathy. Pediatric Neurology. 34 (5), 360-366 (2006).
- van de Looij, Y., Chatagner, A., Huppi, P. S., Gruetter, R., Sizonenko, S. V. Longitudinal MR assessment of hypoxic ischemic injury in the immature rat brain. Magnetic Resonance in Medicine. 65 (2), 305-312 (2011).
- Shibasaki, J., et al. Changes in brain metabolite concentrations after neonatal hypoxic-ischemic encephalopathy. Radiology. 288 (3), 840-848 (2018).
.