Transl. Stroke Res. (2014) 5:647–652 DOI 10.1007/s12975-014-0356-8
REVIEW ARTICLE
Double Cisterna Magna Blood Injection Model of Experimental Subarachnoid Hemorrhage in Dogs Kentaro Mori
Received: 27 February 2014 / Revised: 12 June 2014 / Accepted: 24 June 2014 / Published online: 3 July 2014 # Springer Science+Business Media New York 2014
Abstract Several animal subarachnoid hemorrhage (SAH) models have been proposed to study the etiology and treatment for cerebral vasospasm. We describe the experimental procedures of a canine double-hemorrhage model of SAH and discuss the pathophysiological parameters and occurrence of angiographic delayed cerebral vasospasm using magnetic resonance (MR) imaging and digital subtraction angiography. Autologous blood was injected twice on days 1 and 3 into the cerebellomedullary cistern of 36 female beagles. All animals showed delayed angiographic vasospasm in the vertebrobasilar arteries on day 7. The degree of vasospasm was 29–42 % of the arterial diameter. However, this model showed no symptomatic vasospasm or ischemic changes detected by MR imaging. This animal model can produce reproducible delayed vasospasm without detectable cerebral infarction on MR imaging. This model allows evaluation of the effect of treatment on delayed vasospasm in the same animals. The canine double-hemorrhage model of SAH is suitable for the quantitative and chronological study of delayed angiographic vasospasm, but not for investigating early brain injury and delayed cerebral ischemia. Keywords Subarachnoid hemorrhage . Cerebral vasospasm . Canine double-hemorrhage model . Early brain injury
Introduction The pathogenesis of cerebral vasospasm after subarachnoid hemorrhage (SAH) is not completely understood, and no definitive treatment has been established. Therefore, appropriate animal models of SAH are required to investigate the pathogenesis K. Mori (*) Department of Neurosurgery, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama 359-8513, Japan e-mail: [email protected]
and to develop new treatments. Various experimental SAH models using the mouse, rat, rabbit, cat, dog, and monkey have been advocated. Various blood injection modalities such as manual blood injection into the cerebral cisterns and arterial perforation have also been reported. Each model has advantages and disadvantages to simulate human SAH caused by rupture of cerebral aneurysm. Which model should be chosen depends on the purpose of the investigation. We used a canine doublehemorrhage model of experimental SAH to develop a new treatment modality for vasospasm [1, 2]. In this review, we show mainly how to perform the canine double-hemorrhage model to obtain stable angiographic vasospasm and discuss the pathophysiological parameters including the degree of angiographic vasospasm. We also discuss the advantages and disadvantages of this canine model for the study of cerebral vasospasm after SAH.
Historical Background of the Model, Evolution, and Modifications The first reproducible model for vasospasm in the basilar artery (BA) of dogs (beagles of either sex weighting 7– 15 kg) was reported in 1972 [3]. This model is called the single cisterna magna injection model in dogs. Briefly, the dog was anesthetized and the cisterna magna was punctured by a 25-gauge needle to inject 2.0 ml of autologous arterial blood after removal of the same amount of cerebrospinal fluid (CSF). The dog’s head was then tilted downwards for 10 min to allow the injected blood to sediment around the BA. Vertebral angiography was performed by the direct puncture method. This single-injection dog model showed a biphasic pattern of vasospasm in the BA during the acute stage (30 min after injection) and chronic stage (2 to 7 days). The constriction rate of the BA diameter was 25 to 59 % (mean 37 %) in the chronic stage. To obtain more consistent and severe chronic vasospasm in dogs, two further hemorrhage
648
canine models were developed by the same group [4, 5]. Briefly, mongrel dogs weighing 16–20 kg were intubated and anesthetized, and 4 ml of autologous arterial blood was injected into the cisterna magna after removal of the same amount of CSF. The cisterna magna blood injection was performed on day 1 and day 3 (48 h later). After each injection, the dog’s head was tilted down for 30 min. Angiographic vasospasm was observed in the chronic stage (day 7) of this double-injection model, which was irreversible by intravenous aminophylline and nifedipine or intraarterial papaverine administration [4]. Such intractable constriction observed in this double-injection model resembles that found in patients after aneurysmal SAH. Chiasmatic cistern manual blood injection models have also been reported using dogs [6, 7]. This model requires percutaneous needle puncture of the suprachiasmatic cistern, and the SAH blood distribution pattern is similar to that caused by anterior circulating artery aneurysms. Arterial bleeding models in dogs have used placement and then withdrawal of the needle with thread on either the internal carotid artery or posterior communicating artery after craniotomy [8, 9]. These models cause arterial bleeding similar to that caused by aneurysm rupture and results in abrupt increase in intracranial pressure. This arterial rupture model also shows the biphasic angiographic vasoconstriction pattern as the cisterna magna blood injection model [8]. These canine SAH models are similar to human SAH following aneurysm rupture but require blind needle puncture in the deeply situated suprachiasmatic cistern or subtemporal craniotomy. Fig. 1 Photographs illustrating the experimental procedures of cisterna magna blood injection using a stuffed toy dog. a Positioning of the dog on a pillow. b Cisterna magna puncture using a 20-gauge needle and flexion of the dog’s neck by hand. c Radiograph showing the puncture point at 3 cm below the inion. d Cerebrospinal fluid is drained by gravity flow. e Autologous nonheparinized blood is injected into the cisterna magna
Transl. Stroke Res. (2014) 5:647–652
Animals Used Either beagle or mongrel dogs of either sex are acceptable for the experimental SAH model. Previous SAH experiments in the canine model found no sex difference in the results [3]. In our institution, female beagles were used, simply because the price of a female dog (approximately US$1,000 per dog) is cheaper than that of male dogs. The beagles were purchased from a laboratory animal breeding and supply company (Kitayama Labs Co., Ltd., Ina, Nagano, Japan). The dogs were 11 to 15 months old (mean 12 months old) and weighed 9 to 11 kg (mean 10 kg). A dog weighing 10 kg is large enough for experimental catheter angiography and magnetic resonance (MR) imaging similar to clinical procedures in humans.
Anesthesia, Perioperative Care, and Monitoring The subcutaneous vein was used for drug administration. The dogs were anesthetized using an intravenous bolus injection of 20 mg/kg of pentobarbital on day 1. The anesthesia was maintained with continuous intravenous infusion of 1 ml/kg/ h propofol. Muscular relaxation was achieved by intravenous injection of 1 ml of vecuronium bromide, which was repeated every 30 to 60 min until the end of the procedure. The dogs were intubated using a 6F tracheal cannula, and respiration was controlled with a MR imaging-compatible mobile
Transl. Stroke Res. (2014) 5:647–652
649
Fig. 2 T1- and T2-weighted and FLAIR MR images on day 7 after the cisterna magna double injection. The lateral ventricle shows slight dilation but no ischemic change was detected
respirator (Pneupac® ParaPAC® Ventilator, Smiths Medical). A 4F double-lumen sheath catheter was placed in the femoral artery for cerebral angiography, blood pressure monitoring, and arterial gas sampling. A rectal thermometer was placed in the rectum and the body temperature maintained around 37 °C. The end-tidal CO2 was monitored to maintain the PaCO2 between 35 and 45 mmHg. T1- and T2-weighted and fluid-attenuated inversion recovery (FLAIR) MR imaging were performed using a 1.5-T MR imaging scanner (Gyroscan 1.5T, Philips Healthcare). The Evans index was calculated from the MR images. At 1.5 h after induction of anesthesia, the left vertebral artery (VA) was cannulated with a 4F catheter under fluoroscopic control, and digital subtraction angiography (DSA) was performed after injection of 3 ml of iopamidol (Bayer Schering Pharma) using an automatic injector (2 ml/s). Arterial diameters were measured using DSA and automatically calibrated for conversion between millimeters and pixels. The diameters of the BA, VA, and superior cerebellar artery (SCA) were quantitatively measured. The BA diameters were measured at three equidistant points between the junctions with the VA and the SCA. The mean of the three measured diameters was taken as the arterial diameter of the BA. The mean of the two measured diameters of the bilateral VAs and SCAs at the junction with the BA were taken as the diameters of the VA and SCA. Arterial blood collected from the double-lumen sheath was analyzed for hemoglobin, hematocrit, pH, PaCO2, PaO2, and serum electrolytes (Na+, K+, Cl−, Ca2+, Mg2+) using a blood gas analyzer equipped with ion-selective electrodes (StatProfile CCX, NOVA Biomedical).
SAH Induction, Technical Considerations, and Angiographic Measurement of Arterial Diameter Cerebral vasospasm was induced by experimental SAH using the canine double-hemorrhage model [4, 5]. Briefly, after the control baseline vertebral angiograms were obtained, the
cisterna magna was punctured using a 20-gauge needle. The dog was laid on a pillow (Fig. 1a). During cisterna magna puncture, the dog’s head was kept in flexion by hand to widen the cranio-cervical junction (Fig. 1b). The puncture point was 3 cm below the inion (Fig. 1c). First, 0.3 ml/kg CSF was
Table 1 Summary of physiological parameters before SAH (day 1) and after SAH (day 7) Parameters
Day 1
Day 7
p value
Body weight (kg) Mean blood pressure (mmHg) Heart rate (/min) Body temperature (°C)
10.3±0.6 123±18 133±30 38.3±0.5
9.5±0.6 101±13 92±26 38.3±0.4
REVIEW ARTICLE
Double Cisterna Magna Blood Injection Model of Experimental Subarachnoid Hemorrhage in Dogs Kentaro Mori
Received: 27 February 2014 / Revised: 12 June 2014 / Accepted: 24 June 2014 / Published online: 3 July 2014 # Springer Science+Business Media New York 2014
Abstract Several animal subarachnoid hemorrhage (SAH) models have been proposed to study the etiology and treatment for cerebral vasospasm. We describe the experimental procedures of a canine double-hemorrhage model of SAH and discuss the pathophysiological parameters and occurrence of angiographic delayed cerebral vasospasm using magnetic resonance (MR) imaging and digital subtraction angiography. Autologous blood was injected twice on days 1 and 3 into the cerebellomedullary cistern of 36 female beagles. All animals showed delayed angiographic vasospasm in the vertebrobasilar arteries on day 7. The degree of vasospasm was 29–42 % of the arterial diameter. However, this model showed no symptomatic vasospasm or ischemic changes detected by MR imaging. This animal model can produce reproducible delayed vasospasm without detectable cerebral infarction on MR imaging. This model allows evaluation of the effect of treatment on delayed vasospasm in the same animals. The canine double-hemorrhage model of SAH is suitable for the quantitative and chronological study of delayed angiographic vasospasm, but not for investigating early brain injury and delayed cerebral ischemia. Keywords Subarachnoid hemorrhage . Cerebral vasospasm . Canine double-hemorrhage model . Early brain injury
Introduction The pathogenesis of cerebral vasospasm after subarachnoid hemorrhage (SAH) is not completely understood, and no definitive treatment has been established. Therefore, appropriate animal models of SAH are required to investigate the pathogenesis K. Mori (*) Department of Neurosurgery, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama 359-8513, Japan e-mail: [email protected]
and to develop new treatments. Various experimental SAH models using the mouse, rat, rabbit, cat, dog, and monkey have been advocated. Various blood injection modalities such as manual blood injection into the cerebral cisterns and arterial perforation have also been reported. Each model has advantages and disadvantages to simulate human SAH caused by rupture of cerebral aneurysm. Which model should be chosen depends on the purpose of the investigation. We used a canine doublehemorrhage model of experimental SAH to develop a new treatment modality for vasospasm [1, 2]. In this review, we show mainly how to perform the canine double-hemorrhage model to obtain stable angiographic vasospasm and discuss the pathophysiological parameters including the degree of angiographic vasospasm. We also discuss the advantages and disadvantages of this canine model for the study of cerebral vasospasm after SAH.
Historical Background of the Model, Evolution, and Modifications The first reproducible model for vasospasm in the basilar artery (BA) of dogs (beagles of either sex weighting 7– 15 kg) was reported in 1972 [3]. This model is called the single cisterna magna injection model in dogs. Briefly, the dog was anesthetized and the cisterna magna was punctured by a 25-gauge needle to inject 2.0 ml of autologous arterial blood after removal of the same amount of cerebrospinal fluid (CSF). The dog’s head was then tilted downwards for 10 min to allow the injected blood to sediment around the BA. Vertebral angiography was performed by the direct puncture method. This single-injection dog model showed a biphasic pattern of vasospasm in the BA during the acute stage (30 min after injection) and chronic stage (2 to 7 days). The constriction rate of the BA diameter was 25 to 59 % (mean 37 %) in the chronic stage. To obtain more consistent and severe chronic vasospasm in dogs, two further hemorrhage
648
canine models were developed by the same group [4, 5]. Briefly, mongrel dogs weighing 16–20 kg were intubated and anesthetized, and 4 ml of autologous arterial blood was injected into the cisterna magna after removal of the same amount of CSF. The cisterna magna blood injection was performed on day 1 and day 3 (48 h later). After each injection, the dog’s head was tilted down for 30 min. Angiographic vasospasm was observed in the chronic stage (day 7) of this double-injection model, which was irreversible by intravenous aminophylline and nifedipine or intraarterial papaverine administration [4]. Such intractable constriction observed in this double-injection model resembles that found in patients after aneurysmal SAH. Chiasmatic cistern manual blood injection models have also been reported using dogs [6, 7]. This model requires percutaneous needle puncture of the suprachiasmatic cistern, and the SAH blood distribution pattern is similar to that caused by anterior circulating artery aneurysms. Arterial bleeding models in dogs have used placement and then withdrawal of the needle with thread on either the internal carotid artery or posterior communicating artery after craniotomy [8, 9]. These models cause arterial bleeding similar to that caused by aneurysm rupture and results in abrupt increase in intracranial pressure. This arterial rupture model also shows the biphasic angiographic vasoconstriction pattern as the cisterna magna blood injection model [8]. These canine SAH models are similar to human SAH following aneurysm rupture but require blind needle puncture in the deeply situated suprachiasmatic cistern or subtemporal craniotomy. Fig. 1 Photographs illustrating the experimental procedures of cisterna magna blood injection using a stuffed toy dog. a Positioning of the dog on a pillow. b Cisterna magna puncture using a 20-gauge needle and flexion of the dog’s neck by hand. c Radiograph showing the puncture point at 3 cm below the inion. d Cerebrospinal fluid is drained by gravity flow. e Autologous nonheparinized blood is injected into the cisterna magna
Transl. Stroke Res. (2014) 5:647–652
Animals Used Either beagle or mongrel dogs of either sex are acceptable for the experimental SAH model. Previous SAH experiments in the canine model found no sex difference in the results [3]. In our institution, female beagles were used, simply because the price of a female dog (approximately US$1,000 per dog) is cheaper than that of male dogs. The beagles were purchased from a laboratory animal breeding and supply company (Kitayama Labs Co., Ltd., Ina, Nagano, Japan). The dogs were 11 to 15 months old (mean 12 months old) and weighed 9 to 11 kg (mean 10 kg). A dog weighing 10 kg is large enough for experimental catheter angiography and magnetic resonance (MR) imaging similar to clinical procedures in humans.
Anesthesia, Perioperative Care, and Monitoring The subcutaneous vein was used for drug administration. The dogs were anesthetized using an intravenous bolus injection of 20 mg/kg of pentobarbital on day 1. The anesthesia was maintained with continuous intravenous infusion of 1 ml/kg/ h propofol. Muscular relaxation was achieved by intravenous injection of 1 ml of vecuronium bromide, which was repeated every 30 to 60 min until the end of the procedure. The dogs were intubated using a 6F tracheal cannula, and respiration was controlled with a MR imaging-compatible mobile
Transl. Stroke Res. (2014) 5:647–652
649
Fig. 2 T1- and T2-weighted and FLAIR MR images on day 7 after the cisterna magna double injection. The lateral ventricle shows slight dilation but no ischemic change was detected
respirator (Pneupac® ParaPAC® Ventilator, Smiths Medical). A 4F double-lumen sheath catheter was placed in the femoral artery for cerebral angiography, blood pressure monitoring, and arterial gas sampling. A rectal thermometer was placed in the rectum and the body temperature maintained around 37 °C. The end-tidal CO2 was monitored to maintain the PaCO2 between 35 and 45 mmHg. T1- and T2-weighted and fluid-attenuated inversion recovery (FLAIR) MR imaging were performed using a 1.5-T MR imaging scanner (Gyroscan 1.5T, Philips Healthcare). The Evans index was calculated from the MR images. At 1.5 h after induction of anesthesia, the left vertebral artery (VA) was cannulated with a 4F catheter under fluoroscopic control, and digital subtraction angiography (DSA) was performed after injection of 3 ml of iopamidol (Bayer Schering Pharma) using an automatic injector (2 ml/s). Arterial diameters were measured using DSA and automatically calibrated for conversion between millimeters and pixels. The diameters of the BA, VA, and superior cerebellar artery (SCA) were quantitatively measured. The BA diameters were measured at three equidistant points between the junctions with the VA and the SCA. The mean of the three measured diameters was taken as the arterial diameter of the BA. The mean of the two measured diameters of the bilateral VAs and SCAs at the junction with the BA were taken as the diameters of the VA and SCA. Arterial blood collected from the double-lumen sheath was analyzed for hemoglobin, hematocrit, pH, PaCO2, PaO2, and serum electrolytes (Na+, K+, Cl−, Ca2+, Mg2+) using a blood gas analyzer equipped with ion-selective electrodes (StatProfile CCX, NOVA Biomedical).
SAH Induction, Technical Considerations, and Angiographic Measurement of Arterial Diameter Cerebral vasospasm was induced by experimental SAH using the canine double-hemorrhage model [4, 5]. Briefly, after the control baseline vertebral angiograms were obtained, the
cisterna magna was punctured using a 20-gauge needle. The dog was laid on a pillow (Fig. 1a). During cisterna magna puncture, the dog’s head was kept in flexion by hand to widen the cranio-cervical junction (Fig. 1b). The puncture point was 3 cm below the inion (Fig. 1c). First, 0.3 ml/kg CSF was
Table 1 Summary of physiological parameters before SAH (day 1) and after SAH (day 7) Parameters
Day 1
Day 7
p value
Body weight (kg) Mean blood pressure (mmHg) Heart rate (/min) Body temperature (°C)
10.3±0.6 123±18 133±30 38.3±0.5
9.5±0.6 101±13 92±26 38.3±0.4
