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 birth^5,6,7,8,9. 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 imbalance^4,5. 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 artery^12,13. 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

All the experimental procedures were approved by the Oklahoma Medical Research Foundation (OMRF) Institutional Animal Care and Use Committee (protocol IACUC #17-17). Pregnant female Sprague-Dawley rat pups at E14 were used for the present study. The animals were obtained from a commercial source (see Table of Materials).

1. Animal preparation

1. Acclimatize the animals in the animal facility prior to delivery of the litters.
2. Maintain all rats on a12 h light/darkcycleand feed standard ratchow.
3. After delivery of the litters, keep the pups with their respective dams. Use both sexes for the experiments.
4. At postnatal age 10 (P10), randomize the pups to either sham or HIE groups.
   ​NOTE: The experimental timeline is depicted in Figure 1. All the experiments were performed on the same day at P10.

2. Carotid artery ligation (CAL) for the experimental HIE group

1. Place the rat pups on a warming pad.
2. Initiate anesthesia with 4% isoflurane in oxygen (0.6 LPM) using a nasal cone until the pinch reflex disappears. Drop the flow of gas to 0.5%-2% for the maintenance of anesthesia. Ensure the pups are unconscious without suppressing the respiratory drive.
3. Mark the pups on the tail for identification and gently restrain them with tape on all four limbs.
4. Shave the neck area and sterilize with 70% iodine-povidone solution swabs (see Table of Materials).
5. Using an #11 blade, make a 1 cm midline neck incision through the skin. Carefully dissect the left parotid gland and fascia until the left carotid artery is exposed. Gently mobilize the vessel using hemostats to free it from the fascia.
6. Using a small hemostat, carefully pass two 5-0 sutures (see Table of materials) around the vessel, 0.5 cm apart, and tie them tightly.
7. Using small scissors, cut the artery in between the two sutures to ensure the discontinuity of blood flow. Close the skin and fascia with 5-0 silk sutures (see Table of Materials).
8. Inject buprenorphine (10 units in 800 μL of sterile normal saline) intraperitoneally and another 800 μL of normal saline subcutaneously in the back of the neck to prevent dehydration.
   NOTE: The procedure must be completed within 10-12 min.
9. Return the pups to the cages with their dams and allow the pups to awaken and recover for 1-2 h.

3. Sham surgical procedure for the control group

1. Place the rat pups on a warming pad.
2. Initiate anesthesia with 4% isoflurane in oxygen (0.6 LPM) using a nasal cone until the pinch reflex disappears. Drop the flow of gas to 0.5%-2% for the maintenance of anesthesia. Ensure the pups are unconscious without suppressing the respiratory drive.
3. Mark the pups on the tail for identification and gently restrain them with tape on all four limbs.
4. Shave the neck area and sterilize with 70% iodine-povidone solution swabs.
5. Using an #11 blade, make a 1 cm midline neck incision through the skin and then close it with 5-0 silk sutures.
6. Follow the same hydration, analgesia, and postoperative care as for the HI group (steps 2.8-2.9).

4. Hypoxia exposure for both the CAL and sham groups

1. Prepare the clear plexiglass hypoxia chamber (see Table of Materials) by attaching tubing to the chamber lid to provide continuous airflow of 6 LPM of the hypoxia gas mixture (8% oxygen, 92% nitrogen).
2. Place a blue absorbent pad in the chamber and immediately place the rat pups from both groups in the chamber. Allow the pups to remain in the hypoxia chamber for 45 min.
3. Immerse the chamber in a water bath with continuously flowing warm water to keep the temperature set at 37 °C inside the chamber.
4. Hydrate the pups with an oral saline solution via gavage of 600 μL before being placed in the hypoxia chamber and 600 μL at the end of the 45 min.
5. Remove all the pups to the cages with their dams (experimental and sham) and allow them to recover for 2 h in a room next to the small animal imaging facility.

5. Magnetic resonance imaging and spectroscopy

1. Perform MRI and MRS to identify and evaluate the radiological and metabolic markers 4 h after the end of carotid artery ligation. Perform the procedure under anesthesia with continuous cardiovascular monitoring at the small animal imaging facility.
2. Anesthetize each animal (with 1.5% isoflurane and 0.7 L/min oxygen) and place it in the MR probe (see Table of Materials) in a supine position on a blue absorbent pad covering a heating pad. Monitor the respiration rate of the animals continuously using an abdominal pneumatic pillow (see Table of Materials).
3. Use a head surface coil as a signal receiver and transmit radiofrequency pulses to the sample through a quadrature volume coil (72 mm inner diameter, see Table of Materials).
4. Perform MRI to evaluate both the changes in cerebral blood flow (CBF) and the changes in water diffusion constants (ADC) following previously published methods^15,16,17. Perform MRI morphology (T1 and T2), diffusion, and perfusion to determine the brain's most affected and least perfused areas.
   NOTE: The mean values for perfusion and diffusion (ADC) in each group are compared between the ligated side and the control side (intact carotid artery side).
5. Perform MRS following previously published methods^15,17 and analyze using an in-house coded program using Mathematica software (see Table of Materials).
6. Scale the MR spectra in parts per million (ppm) by calibrating against the water peak (4.78 ppm). Identify the major brain metabolic peaks as N-acetylaspartate (NAA) at 2.02 ppm, choline (Cho) at 3.22 ppm, creatine (Cr) at 3.02 ppm, and myo-inositol at 3.53 ppm.
   ​NOTE: The peak area measurements of the metabolites are used to calculate the following ratios: NAA to Cho (NAA/Cho), Cr to Cho (Cr/Cho), and Myo-Ins to Cho (Myo-Ins/Cho)¹⁵.

6. Serum and cerebral tissue analysis

1. Perform blood sampling at 5.5 h following the carotid artery ligation or sham procedure according to previously published methods¹⁸.
2. Anesthetize the pups again with 4% isoflurane.
3. Using a sharp #11 blade, make an abdominal incision, followed by a diaphragmatic incision to expose the heart.
4. Perform blood sampling via cardiac puncture as previously described¹⁸. Briefly, insert a 32 G needle on a 1 mL syringe into the right heart chamber and gently aspirate 1 mL of blood.
5. Allow the whole blood to coagulate, followed by centrifugation at 1,000 x g for 15 min at 4 °C. The serum gets separated into clean microcentrifuge tubes.
6. Decapitate the whole pup head for the gross assessment of cerebral pathology and then immerse it in ice for 2 min.
7. Make an incision on the dorsal scalp from the base of the skull to the tip of the nose and peel the skull bones from around the brain. Remove the intact whole brain into a clean Petri dish.
8. Mark the right side of the brain with a non-toxic marker. Position the brain with the cephalad surface upward so that both hemispheres are visible. Using an ice-cold razor blade, slice the brain into four equal sections parallel to the coronal plane.
9. Immerse the brain sections in 2,3,5-triphenyltetrazolium chloride (TTC, see Table of Materials) solution in a Petri dish covered with foil to prevent photosensitization and incubate for 15 min at 37 °C.
   NOTE: Infarcted areas are delineated as white areas devoid of red TTC stain.
10. If infarcts are subtle or difficult to detect, inject 0.5-1 mL of TTC (1%) in phosphate-buffered saline directly into the right heart after abdominal incision and thoracotomy, and allow to perfuse for 2 min prior to the decapitation of the pup.
11. Store the brain tissue and serum at −80 °C if further analysis is required.

Results

The present protocol to produce and evaluate early cerebral changes after HIE was easy to implement and allowed gross pathological and radiological visualization of cerebral injury within 6 h of insult in rat pups at P10. The experimental plan is depicted in Figure 1. Both sexes were analyzed together, and 24 animals from five litters were examined in each group. Animal mortality was very low, with 99% survival of animals until the terminal experiments were performed.

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...

Materials

Name	Company	Catalog Number	Comments
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