- Department of Neurosurgery, UCLA David Geffen School of Medicine, Los Angeles, CA, USA
- Department of Radiology, UCLA David Geffen School of Medicine, Los Angeles, CA, USA
- Department of Neurosurgery and Radiology, UCLA David Geffen School of Medicine, Los Angeles, CA, USA
- Department of Surgery, West Los Angeles Veteran's Administration, Los Angeles, CA, USA
Nestor R. Gonzalez
Department of Neurosurgery and Radiology, UCLA David Geffen School of Medicine, Los Angeles, CA, USA
DOI:10.4103/2152-7806.83023Copyright: © 2011 Dusick JR. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
How to cite this article: Dusick JR, Evans BC, Laiwalla A, Krahl S, Gonzalez NR. A minimally-invasive rat model of subarachnoid hemorrhage and delayed ischemic injury. Surg Neurol Int 18-Jul-2011;2:99
How to cite this URL: Dusick JR, Evans BC, Laiwalla A, Krahl S, Gonzalez NR. A minimally-invasive rat model of subarachnoid hemorrhage and delayed ischemic injury. Surg Neurol Int 18-Jul-2011;2:99. Available from: http://sni.wpengine.com/surgicalint_articles/a-minimally-invasive-rat-model-of-subarachnoid-hemorrhage-and-delayed-ischemic-injury/
Background:Double-injection models of subarachnoid hemorrhage (SAH) in rats are the most effective in producing vasospasm, delayed neurological deficits and infarctions. However, they require two large surgeries to expose the femoral artery and the atlanto-occipital membrane. We have developed a minimally-invasive modification that prevents confounding effects of surgical procedures, leakage of blood from the subarachnoid space and minimizes risk of infection.
Methods:Rats are anesthetized and the ventral tail artery is exposed through a small (5 mm), midline incision, 0.2 mL of blood is taken from the artery and gentle pressure is applied for hemostasis. The rat is flipped prone, and with the head flexed to 90 degrees in a stereotactic frame, a 27G angiocath is advanced in a vertical trajectory, level with the external auditory canals. Upon puncturing the atlanto-occipital membrane, the needle is slowly advanced and observed for cerebrospinal fluid (CSF). A syringe withdraws 0.1 mL of CSF and the blood is injected into the subarachnoid space. The procedure is repeated 24 hours later by re-opening the tail incision. At 8 days, the rats are euthanized and their brains harvested, sectioned, and incubated with triphenyltetrazolium chloride (TTC).
Results:Rats develop neurological deficits consistent with vasospasm and infarction as previously described in double-injection models. Cortical and deep infarctions were demonstrated by TTC staining and on histopathology.
Conclusions:A minimally invasive, double-injection rat model of SAH and vasospasm is feasible and produces neurological deficits and infarction. This model can be used to study neuroprotective treatments for vasospasm and delayed neurological deficits following SAH, reducing the confounding effects of surgical interventions.
Keywords: Delayed ischemic deficit, double-injection model, rat, subarachnoid hemorrhage, vasospasm
Several methods have been proposed for the experimental modeling of subarachnoid hemorrhage (SAH) and resulting vasospasm and delayed neurological deficits.[
The double-injection rat model was first presented by Solomon et al.[
We have developed a minimally invasive modification that maintains the reproducibility and effect of the double-injection SAH model while preventing the potentially confounding effects of the surgical procedures, avoiding leakage of blood from the subarachnoid space after injection and minimizing the risk of wound infection.
Male adult Sprague-Dawley rats are anesthetized on Day 1 of the experimental protocol by intraperitoneal injection of midazolam (1 mg/kg) and ketamine (100 mg/kg). This anesthesia is commonly used in rat SAH models, its safety has been proven for imaging procedures in neurologically impaired rats and it has not been shown to cause significant changes in cerebral blood flow.[
The anesthetized rats are placed supine on a heating plate to maintain body temperature at 37°C. Under the microscope, the ventral tail artery is exposed through a small (5 mm), midline incision. Using a 27-gauge needle and heparinized syringe, 0.2 mL of arterial blood is taken from the artery and gentle pressure is applied for hemostasis. The tail incision is then closed.
The rats are then flipped prone and the head is fixed in a stereotactic frame. The head is flexed to 90 degrees to horizontal and secured in this position. A 27-gauge angiocath is secured to a vertically oriented microdrive and is aligned horizontally with the external auditory canals [
This same procedure is repeated 24 hours later (Day 2) by re-opening the tail incision. If need be, the artery can be exposed more proximally to obtain this blood sample. The rats are evaluated daily thereafter and scored on a standard neurological behavioral score.[
On Day 8, the rats are euthanized and their brains immediately harvested. The brains are sectioned coronally into 2 mm slices. These slices are then incubated at 37°C with 2% triphenyltetrazolium chloride (TTC) for 20 minutes for identification of infarcted brain.[
Our Institution's Animal Care Committee approved this protocol.
Results of our initial experience
To date we have performed this technique on two rats. Using the method of evaluation first proposed by Bederson et al.,[
Gross inspection of the harvested brains shows diffuse SAH in the basal cisterns [
(a) Gross pathology of harvested brain on Day 8. Note that the cisternal injection produced accumulation of blood in the prepontine and interpeduncular cisterns (white arrows). (b) Triphenyltetrazolium chloride staining reveals numerous cortical and deep infarctions in the sectioned brain. These infarctions correlated with the neurological deficits observed in the rat prior to euthanasia
Impact and comparison to previous models
Spontaneous SAH from rupture of a cerebral aneurysm is a major health problem.[
A consistently reproducible, easy-to-perform, and cost-effective experimental model to simulate aneurysm rupture and delayed neurological deterioration is invaluable to future investigations into both the pathophysiology of SAH as well as neuroprotective treatments that aim to reduce morbidity and mortality following SAH.[
The one model that has been shown to both consistently produce vasospasm, delayed neurological deficits, and infarction and to be relatively cost-effective, is easy to perform (compared to large animal models) and has a low mortality rate is the rat cisternal double-injection model, which utilizes two injections of arterial blood into the cisterna magna spaced 24 hours apart.[
Among the multiple mechanisms involved, it has been suggested that an inflammatory response and the associated leukocyte–endothelial cell interactions play a significant role in the pathophysiology of cerebral vasospasm.[
In conclusion, a minimally invasive, double-injection rat model of SAH and vasospasm is feasible and produces neurological deficits and infarction as has been previously demonstrated with similar models. This model may be used to study neuroprotective treatments for vasospasm and delayed neurological deficits following SAH, reducing the confounding effects of surgical interventions inherent to most other models.
The authors wish to acknowledge Harry Vinters, MD, for the processing and analysis of the pathological specimens.
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