Awake Rat Model of Ischemic Stroke with Neuropsychiatric Complications During a Seven-Day Observation

Summary

The new model of focal ischemic stroke in awake rats is proposed. This was achieved by injecting an air embolus into the internal carotid artery of rats. The development of focal ischemic stroke was confirmed by brain staining with triphenyltetrazolium chloride, functional tests, and assessment of physiological parameters.

Abstract

The translational model of experimental stroke in rats induced by a gas embolism was created to clarify the features of stroke's development and to evaluate the neuroprotective effect of novel remedies and proven drugs. The moderately time and labor-consuming protocol allows for observing the dynamics of rapid neuron death in awake animals, providing a unique opportunity to assess the acute phase of stroke, including delirium, as well as to monitor autonomic disturbances such as changes in blood pressure, body temperature, and respiratory minute volume. Atmospheric air (100 µL) was administered by infusion through a vascular catheter placed retrogradely into the common carotid artery via the external carotid artery, using an infusion pump. Twenty-four hours after embolization, the animals exhibit stereotypical behavioral, motor deficits, and histopathological differences. By the seventh day of the experiment, a significant improvement in neurological status was observed, and the results in behavioral tests approached those of the control values. At the same time, a transition from neutrophilic to macrophage infiltration is observed in the ischemic lesion. The proposed model can be utilized in preclinical studies, as it reproduces the neuropsychiatric complications of the post-stroke period, along with the corresponding physiological, functional, and histopathological changes.

Introduction

The objective of this research was to develop a model of focal cerebral ischemic stroke in awake rats and to achieve the closest approximation of this model to the clinical aspects of ischemic stroke in humans. As demonstrated in the research by Söyland et al., 62.6% of patients exhibited significant symptoms of ischemic stroke in an awake state, while only 19% experienced a wake-up stroke¹. Consequently, the occurrence of an awake state during cerebral artery embolization in animals is of the greatest interest. The classical models employed for the study of focal cerebral ischemic stroke include the following: transcranial occlusion, endovascular filament middle cerebral artery occlusion, embolic occlusion, endothelin-1 occlusion, and photothrombosis model². Classical modeling methods expose animals to the effects of anesthetics, which may result in the distortion of endpoints in the ischemic stroke model. Isoflurane is extensively employed in occlusion surgery as the most expedient method of gas anaesthesia^3,4,5. However, the degree of cerebral impairment following middle cerebral artery occlusion (MCAO) after 24 h was found to be inversely proportional to the duration of isoflurane anesthesia in animals. It has been demonstrated that isoflurane exerts an effect on over 1,000 ischemia-related differentially expressed genes expression⁶, a factor which may result in an ischemic stroke model that is not entirely reliable.

One methodology of ischemic stroke modeling in rats is the injection of the blood clot into the internal carotid artery⁷. Nevertheless, the induction of ischemic stroke in this model was performed 1 h after catheterization with isoflurane. It is conceivable that this preconditioning may have disturbed the development of the neurological and physiological consequences of thrombus administration.

The alternative method for modeling ischemic stroke by embolization of the internal carotid artery in awake rats, followed by focal brain damage in the middle cerebral artery basin, is proposed. The catheterization was performed 24 h prior to the modeling of the ischemic stroke. This ensured that the effects of the embolus administration were not affected by the anesthesia.

The proposed model does not utilize endothelin-1⁸, filament⁹, or coagulable pigment¹⁰ for ischemia. The selection of the agent to be administered to the rats via a catheter was based on the premise that this method would be feasible, thus obviating the necessity of fixing the animal in a stereotaxic frame and anesthetizing it^8,10.

Furthermore, the model enables the observation of physiological and neurological parameters in animals immediately after embolization; alternative methods of ischemic stroke modeling appear to be incompatible with this. In the literature, the endpoints of brain damage and neurological impairment after ischemic stroke modeling are most often assessed 24 h after occlusion^11,12,13,14.

The utilization of awake rats in ischemic stroke modeling has the potential to serve as an additional or alternative to the prevailing classical model of focal ischemic stroke. This approach facilitates a more extensive investigation of the neuroprotective potential of registered or developing pharmaceutical agents for the treatment of ischemic stroke.

Protocol

The study was conducted in accordance with the standard operating procedures of the laboratory and was approved by the Bioethical Commission of the Institutional Animal Care and Use Committee of the Branch of the Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences (Protocol No. 1016/25, dated January 17, 2025). In this study, 28 male Sprague-Dawley (SD) rats, aged 10-12 weeks, were used, with an average body weight of 301 g and a microbiological status of Specific Pathogen Free (SPF). The reagents and the equipment used are listed in the Table of Materials.

1. Animal preparation

1. Use 28 male SD rats, 10-12 weeks old, with SPF status. Use 12 animals for the sham group, and 16 animals to model ischemic stroke with cerebral arterial gas embolism (CAE).
2. Sterilize all instruments for 24 h in the disinfectant solution (0.5% chlorhexidine bigluconate in 70% ethanol).
3. Anesthetize rats by intramuscular injection with a mixture of tiletamine 15 mg/kg and zolazepam 15 mg/kg + xylazine hydrochloride 10 mg/kg (following institutionally approved protocols).
4. Ensure that the surgical stage of anesthesia has been achieved: touch the corner of the animal's eye, pinch the tip of the animal's tail.
   NOTE: The animal is considered anesthetized when no reflexes are observed after these manipulations.
5. Pluck the operating field on the ventral neck region.
6. Fixate the animal on the surgical table by fixing the limbs and upper incisors with soft tourniquets.
7. Cut polyethylene tubes 0.8 mm in diameter into 10 cm long tubes, then twist a stabilizing spiral about 5 mm wide at the tip by threading the tip of the polyethylene tube onto a metal tube and dipping it in hot water (90 °C). Fill the tube with heparin (50 ME). Seal the opposite end of the spiral with a plug made from a piece of fishing line.
   NOTE: The catheter is made out of medical-grade polyethylene tubes. Avoid kinking or pinching the catheter when handling it.

2. Carotid artery catheterization

1. Make an incision about 5 mm long on the ventral side of the animal's neck.
2. Dissect the neck muscle using forceps. Open the area where the carotid artery branches into the external and internal carotid arteries.
3. Find the artery running along the trachea. Carefully release it with forceps from the connective tissue sheath without touching the vagus nerve running along the artery.
4. Use forceps to isolate a section of the external carotid artery.
5. Place a temporary ligature 3-5 mm caudal to the bifurcation on the common carotid artery.
6. Place two ligatures on the external carotid artery, one immediately after the bifurcation and one distal 4 mm from the bifurcation.
   NOTE: Catheter insertion will be performed between these two ligatures. The ligature placed immediately after the bifurcation will fixate the catheter in the vessel.
7. On the internal carotid artery, place one temporary ligature cranial to the bifurcation of the common carotid artery.
8. Place a ligature on the pterigopalatina artery after the bifurcation.
   NOTE: When applying the ligatures in steps 2.7 and 2.8, bleeding may occur due to the presence of collaterals hidden behind the main trunk of the common carotid artery. In this case, they should be ligated.
9. Make an incision on the external carotid artery between the ligatures using scissors. Insert the prepared catheter (step 1.7) into the hole formed in the vessel, guiding it towards the common carotid artery.
10. Release the distal ligature and push the catheter further down the vessel until it enters the common carotid artery. Carefully tie two ligatures on the section of the external carotid artery with the catheter inside. Place the third ligature before the catheter, and tie it as well.
    1. Fixate the catheter with ligatures to the cushioning ring. Remove the ligatures from the carotid and external carotid arteries.
       NOTE: When pushing the catheter, blood may leak from the vessel.
11. In the case of blood leaking from the vessel, place duplicate ligatures between the hole in the vessel and the third ligature.
12. Guide the fixed catheter to the withers with a grooved needle director.
13. Suture the ventral wound with 4.0 suture material. Use a continuous suture.
14. Suture the dorsal side of the skin with 4.0 suture material tightly around the catheter. Use a continuous suture. Attach a piece of adhesive plaster to the catheter exterior and tie it to the suture for a more secure fixation.
15. Administer the antibiotic ciprofloxacin at a dose of 5 mg/kg intramuscularly.

3. Focal cerebral ischemia modeling in the CAE group animals

1. Twenty-four hours after rat catheterization, place the animal in an awake state in the lab animal immobilizer so that it is possible to access the catheter placed on the withers.
2. Place a syringe filled with 0.5 mL saline solution (0.9% NaCl) with a 30 G needle into the infusion pump.
3. Connect the syringe needle to an empty polyethylene catheter (100 µL capacity). Connect the other end of the polyethylene catheter to the implanted catheter using an adapter.
   NOTE: The proper catheter implantation and the absence of a thrombus are proven when heparin from the implanted catheter is transferred into the catheter containing air, causing the air to be slightly compressed. The reason for this is that the blood pressure of the rat exceeds the pressure of the air in the connected catheter.
4. Set the injection rate on the infusion pump to 10 µL/min. Set the injection volume on the infusion pump to 120 µL.
5. Start the infusion pump. Inject 100 µL of air from the polyethylene catheter and 20 µL of saline solution (0.9% NaCl).
   NOTE: The injection is a 10 min process. At this time, observe the animal. During injection, the animal can vocalize, gnaw, or dig the immobilizer.
6. Take the animal out of the immobilizer.
7. Immediately assess the animal's neurological condition using a 0-5 point scale: 0 = no neurological deficit, 1 = inability to fully straighten the forelimb, 2 = rotation, 3 = loss of limb support, 4 = no spontaneous walking with depressed level of consciousness, and 5 = death.
   NOTE: For a more detailed description, refer to source¹⁵. It's possible that an animal will not exhibit neurological impairment immediately after the air is administered. This indicates that occlusion did not occur due to the animal's anatomical features or elevated blood pressure.
8. Exclude the animal from the experiment in the absence of neurological symptoms.
   NOTE: Rats with CAE demonstrate 1-4 points on the neurological deficit scale. Most often, it is loss of limb support, or no spontaneous walking with a depressed level of consciousness.
9. Administer the antibiotic ciprofloxacin at a dose of 5 mg/kg intramuscularly.

4. Assessment of physiological parameters

1. One hour after CAE, assess the animal's neurological condition using a 0-5 point scale: 0 = no neurological deficit, 1 = inability to fully straighten the forelimb, 2 = rotation, 3 = loss of limb support, 4 = no spontaneous walking with depressed level of consciousness, and 5 = death¹⁵.
   NOTE: After 1 h, the animal may not show any symptoms due to the air bubble being reabsorbed rapidly, indicating that the arterial embolization was insufficient.
2. Exclude the animal from the experiment in the absence of neurological symptoms 1 h after CAE (rats with 0 points on the neurological deficit scale¹⁵).
3. Administer the antibiotic ciprofloxacin at a dose of 5 mg/kg intramuscularly at 24 h and 48 h after CAE.
4. Randomize animals from the sham group and CAE group for assessment of physiological parameters.
5. Weigh animals immediately before CAE, and 3 h, 24 h, 4 days, and 7 days after CAE on a Sartorius scale with gram accuracy.
6. Measure the rectal body temperature of rats immediately before CAE, and 3 h, 24 h, 4 days, and 7 days after CAE using a digital thermometer with an operating range of 32 °C to 42 °C.
7. Measure systolic blood pressure and heart rate noninvasively immediately before CAE, and 3 h, 24 h, 4 days, and 7 days after CAE using a tail cuff on a computerized system to record blood pressure and respiratory parameters.
   1. Turn on the heating pad and heat it to a temperature of 40 °C.
   2. Place the animal in the animal immobilizer and position it on the heating pad. Allow the animal to calm down for 3-5 min.
   3. Then, place a cuff with a sensor for measuring cardiovascular parameters on the tail of the animal, 2-3 cm from the base of the tail.
   4. Adjust the transducer placement directly under the ventral tail artery.
   5. Ensure that the signal is stable and the heart rate is displayed in the program. Start recording the signal.
   6. Turn on the cuff pressure build-up.
   7. Record the pulse and systolic blood pressure until the cuff pressure is fully released.
   8. Ensure that the recovery of the animal's heart rate signal is clearly displayed.
   9. Repeat steps 4.4.3-4.4.5. Two more times and release the animal from the animal immobilizer.
   10. Calculate the average pulse and systolic blood pressure from three measurements.
8. Measure respiratory rate and respiratory volume immediately before CAE, and 3 h, 24 h, 4 days, and 7 days after CAE using a computerized system to record blood pressure and respiratory parameters.
   1. Secure the animal in one hand in a natural position for the animal.
   2. Place a mask with a tube connected to the animal's nose.
      NOTE: It will take time for the animal to get used to breathing into the mask.
   3. Ensure that the animal is calmed down.
   4. Record respiratory volume and respiratory rate for 10 s.
   5. Ensure that the animal did not sigh or swallow during the recording.
   6. Repeat steps 4.5.2-4.5.3 two more times.
   7. Calculate the average respiratory volume and respiratory rate from three measurements.
   8. Calculate the respiratory minute volume using the formula:
      Minute respiratory volume = Respiratory rate × Respiratory volume.

5. Assessment of functional and behavioral parameters

1. Assess the animal's neurological condition using a 0-5 point scale: 0 = no neurological deficit, 1 = inability to fully straighten the forelimb, 2 = rotation, 3 = loss of limb support, 4 = no spontaneous walking with depressed level of consciousness, and 5 = death¹⁵.
2. Assess motor function and coordination on a Rota-Rod immediately before CAE, and 3 h, 24 h, 4 days, and 7 days after CAE.
   1. Train the animals for testing on the Rota Rod device.
      1. Place the animal on a stationary rod for 5-30 s.
      2. Animals capable of holding on to a stationary rod for at least 10 s, have them run on a rod rotating with minimal acceleration for about 30 s.
      3. Continue training the animals until they are able to stand freely on a rod rotating at an appropriate acceleration.
   2. Place the animals on a 7 cm diameter rotating horizontal rod at a minimum speed of 1 rpm.
   3. Start acceleration of 1 rpm every 10 s up to a maximum speed of 60 rpm.
   4. Record the time the rat has been on the rotating rod.
3. Assess rat locomotor activity immediately before CAE, and 3 h, 24 h, 4 days, and 7 days after CAE using a computerized system.
   1. Place the animal in the center of an open field and record for 5 min the distance traveled and the rat's activity time.
4. Assess forelimb grip strength 3 h, 24 h, 4 days, and 7 days after CAE on the Grip Strength Meter.
   1. Hold the animal by the tail and allow the animal to grasp the bars with its forelimbs.
   2. Pull the animal parallel to the grate until the rat releases the grate.
   3. Make three attempts and select the highest result.

6. Assessment of S100 calcium-binding protein B (S100b) level

1. Measure serum S100b levels in rats before CAE modeling, 24 h, and 7 days after CAE.
   1. Place the animal in the laboratory animal immobilizer.
   2. Turn on the heating pad and heat it to a temperature of 40 °C.
   3. Position the animal in the laboratory animal immobilizer on the heating pad. Wait for a period of 5 min in order to allow the animal to calm down.
   4. Place the animal on its side.
      NOTE: The caudal vein should be visible on the tail.
   5. Insert an intravenous catheter into the caudal vein.
   6. Draw 1 mL of blood into a 1.5 mL tube.
   7. Let the blood stand at room temperature for a period of 40 min.
   8. Centrifuge the blood sample at 4 °C for 15 min at 1500 x g.
   9. Collect the serum with an automatic pipette into a separate 1.5 mL tube.
      NOTE: The serum must be clear and transparent, without any red admixture of erythrocytes.
   10. Analyze the serum by enzyme-linked immunosorbent assay (ELISA) using an appropriate ELISA kit.

7. Catheter removal

1. Perform the catheter removal procedure 24 h after CAE, after tests in step 5.
   1. Place the animals in the inhalation chamber with a connected gas anesthesia system and oxygen concentrator for 1-2 min with isoflurane supply in the chamber at a volume of 2.5%-3.5% with oxygen supply rate of 3 L/min.
   2. Transfer the animal to a heating pad (37.5 °C) with a mask gas supply with isoflurane 2%-3% dosing mode with oxygen flow rate of 1.0-2.0 L/min.
   3. Cut the flag without cutting the wound suture or damaging the catheter.
   4. Clean the outer end of the catheter, treat with disinfectant solution (0.5% chlorhexidine bigluconate in 70% ethanol).
   5. Remove the suture from the previous surgery from the ventral side of the neck. Through the incision, use forceps to gently isolate the catheter helix.
   6. Pull out the long end of the catheter through the ventral incision.
   7. Place a ligature with a knot, without tightening, on the external carotid artery between the bifurcation and the catheter insertion site.
   8. Carefully cut the knots binding the catheter, pull the catheter out with forceps, tighten the knot on the vessel, and re-tie.
   9. Trim the ligature ends and suture the wound. Use a continuous suture.

8. Assessment of triphenyltetrazolium chloride staining

1. Provide terminal anesthesia to 6 animals from the sham group and 6 animals from the CAE group 24 h after occlusion, and 6 animals from the sham group and 10 animals from the CAE group on the 7th day after CAE using intramuscular injection of tiletamine 15 mg/kg and zolazepam 15 mg/kg + xylazine hydrochloride 10 mg/kg.
2. Verify that the surgical stage of anesthesia has been achieved as described in step 1.3.
3. Perform a total blood sampling from the caudal vena cava with a 2 mL syringe and a 24 G needle.
4. Open the dorsal vault of the skull without damaging the brain tissue.
5. Examine the dura mater.
6. Slice the brain with a microtome blade into 2 mm-thick frontal slices starting from the frontal lobe.
7. Place the sections on a microscope slide.
8. Place the slices in 1% triphenyltetrazolium chloride (TTC) solution and place them in a thermostat at 37 °C for 10-12 min.
9. Examine all brain sections. Observe the shift in color of TTC staining.
   NOTE: Undamaged tissue will stain bright pink, while tissue with lesions will stain pale pink.
10. Photograph the slices from both sides.
11. Fix the slices in 10% neutral buffered formalin solution.
12. Estimate the area of the focal lesion in the software for scientific image analysis.

9. Assessment of hematoxylin and eosin staining

1. Dehydrate fixed brain slices in an ascending battery of ethanol (70%, 80%, 100%).
2. Soak dehydrated slices in paraffin and make slices 7 mm thick.
3. Stain the sections with hematoxylin and eosin.
4. Conduct microscopic analysis of sections using a transmitted light microscope.

10. Statistical analysis

1. Perform statistical analysis in the statistical program.
2. Apply descriptive statistics (calculate the mean ± standard error of the mean), and make graphs to visualize the results.
3. After checking the normality of each group, perform multiple comparisons using Duncan's rank criterion.
4. Determine differences at the 5% significance level.

Results

Survival and overall condition
Immediately after administration of the air bubble, all rats showed a disturbed state and a pronounced tremor for several seconds, followed by cessation of all motor activity and loss of consciousness for 1-2 min. Some animals lost support on their paws for 10-15 min after regaining consciousness.

The survival rate of animals 3 h after CAE was 100%, but after 24 h the survival rate decreased to 80%, and on the 3^rd day it was 60%. A...

Discussion

MCAO is a common method of modeling focal ischemic stroke in rats, which is achieved by thrombus formation, local spasm of the middle cerebral artery, and temporary occlusion by filament². The aim of such models is (1) to achieve focal apoptosis of neural brain tissue¹⁶ with standardized lesion volume and area¹⁷, (2) to monitor the neurological deficit of animals for 2-7 days¹⁸.

The selection of the...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
3-component syringe with a 2 mL needle	LEIKO	523222	23G
Aladdin Single infusion pump	World Precision Instruments	AL-1000
Ciprofloxacin	Pharmavet	3897744	5% solution of ciprofloxacin
Computerized system PowerLab 8/35	ADInstruments	3508-1090
Dezin	Apteka.ru		0.5% chlorhexidine bigluconate in 70% ethanol
Digital thermometer	B.Well Swiss AG	LOT 0413
Electronic dynamometer Grip Strength Meter	BioMed Easy Technologies	007231024415MC01
ELISA Kit for S100 Calcium Binding Protein B (S100B)	CLOUD-CLONE CORP.(CCC)	SEA567Ra	48T
Fishing line			Diameter 0.9 mm
Gas anesthesia system	UGO BASILE	21100
GraphPad statistical software	Prism		v.10
Grooved Needle Director	Fisher Scientific	Catalog no. 11-00	12.7 cm
Heparin	Endopharm. Moscow Endocrine Plant	30122
ImageJ	National Institutes of Health and the Laboratory for Optical and Computational Instrumentation
Insulin syringe	SFM Hospital Products	160303	0.5 mL, U-100, 30G
Intravenous catheter	KD Medical		G24
Isoflurane (Isoflurin)	Vetpharma		100 mL, 1000 mg/kg
Lab animal immobilizer for laboratory animals	Open Science	AE1001-R1
Light microscope	ZEISS		Axio Scope.A1
Light microscopy slides	Thermo Scientific	15442933
Medical grade polyethylene micro tubing	Scientific Commodities Inc.	BB31695-PE/4	Inner diameter 0.76 mm
Medical sterile gauze wipes	New life		45x29 CM
Micro tube	SARSEDT AG & Co. KG	S044378	1.5 mL
Micropipette Eppendorf research plus	Eppendorf		200 µL
Microscope slides	Fisher Scientific	11562203
Microsurgical forceps (curved)	Kazan Medical Instruments Plant	F0106
Microsurgical forceps (straight)	RWD Life Science	F11029-11
Microtome blades	Thermo Scientific	3052835	MX35 Premier+
Multiconditioning System	TSE Systems	131202-10
Neutral buffered formalin	hem	1072024	pH 6,8-7,2
Non-absorbable Surgical Suture Lavsan	Lintex	2022-01
Ophthalmic scissors for iridectomy	CM Instrumente	AJ-410-05
Oxygen concentrator	Armed	YYT32700960	7F-3L
Phosphate buffer	AMRESCO	2120C462
Piptte tips	Servicebio		200 µL
Rats	obtained from the Laboratory Animal Nursery “Pushchino” (www.spf-animals.ru), Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences.
Refrigerated Lab Multifunctional Centrifuge	Eppendorf	5804 R
Rota-Rod Rotamex-5	COLUMBUS INSTRUMENTS	210426
Scissors METZENBAUM	Medstandart-Instrument	MT-H-256
Surgical suture material	REPROMED	114/0042011124	Kaproag 4/0
Surgical table	VetKormTorg	VETBOT STL-1, TD00472
Telazol	Zoetis	672913	50 mg/mL tiletamine and 50 mg/mL zolazepam in 5 mL
Triphenyltetrazolium chloride	Dia-M	TTC1809
Xylazine	NITA-FARM	151	2% xylazine hydrochloride in 50mL