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Neuroscience

Pre-Chiasmatic, Single Injection of Autologous Blood to Induce Experimental Subarachnoid Hemorrhage in a Rat Model

Published: June 18th, 2021

DOI:

10.3791/62567

1Department of Neurosurgery, Rigshospitalet, 2Department of Clinical Experimental Research, Glostrup Research Institute, Rigshospitalet, 3Department of Clinical Medicine, University of Copenhagen, 4Department of Clinical Sciences, Division of Experimental Vascular Research, Lund University, 5Department of Clinical Neuroscience, Karolinska Institutet

Subarachnoid hemorrhage continues to carry a high burden of mortality and morbidity in man. To facilitate further research into the condition and its pathophysiology, a pre-chiasmatic, single injection model is presented.

Despite advances in treatment over the last decades, subarachnoid hemorrhage (SAH) continues to carry a high burden of morbidity and mortality, largely afflicting a fairly young population. Several animal models of SAH have been developed to investigate the pathophysiological mechanisms behind SAH and to test pharmacological interventions. The pre-chiasmatic, single injection model in the rat presented in this article is an experimental model of SAH with a predetermined blood volume. Briefly, the animal is anesthetized, intubated, and kept under mechanical ventilation. Temperature is regulated with a heating pad. A catheter is placed in the tail artery, enabling continuous blood pressure measurement as well as blood sampling. The atlantooccipital membrane is incised and a catheter for pressure recording is placed in the cisterna magna to enable intracerebral pressure measurement. This catheter can also be used for intrathecal therapeutic interventions. The rat is placed in a stereotaxic frame, a burr hole is drilled anteriorly to the bregma, and a catheter is inserted through the burr hole and placed just anterior to the optic chiasm. Autologous blood (0.3 mL) is withdrawn from the tail catheter and manually injected. This results in a rise of intracerebral pressure and a decrease of cerebral blood flow. The animal is kept sedated for 30 min and given subcutaneous saline and analgesics. The animal is extubated and returned to its cage. The pre-chiasmatic model has a high reproducibility rate and limited variation between animals due to the pre-determined blood volume. It mimics SAH in humans making it a relevant model for SAH research.

Non-traumatic subarachnoid hemorrhage (SAH) is a form of stroke, representing around 5% of all cases. The most common cause of non-traumatic SAH is the sudden rupture of an aneurysm (aSAH), which accounts for 85% of SAHs. Other causes include the rupture of an arterio-venous malformation, coagulopathies, and rupture of veins in perimesencephalic hemorrhage1. The incidence rate is 9 per 100,000 person-years with mortality around one in three and another third requiring the support of daily living following SAH2,3.

Following initial stabilization and diagnosis ....

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This procedure is done in accordance with the European Union's Directive 2010/63/EU regarding the protection of animals used for scientific purposes and approved by the Danish Animal Experiments Inspectorate (license no. 2016-15-0201-00940). Surgery is performed using aseptic technique to the widest extent possible, including sterile instruments, gloves, catheters, and sutures. The study used male and female Sprague-Dawley rats weighing 230-350 g, group housed in 12-h light/dark cycle, with constant temperature of 22.......

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Women have an increased risk of aSAH compared to men. Despite this, male rodents are primarily used in experiments due to possible bias from heterogeneity of estrus cycle in females. The representative results presented here are from a recent publication comparing female and male rats, confirming that the model produces similar results in female animals compared to male21. The study included 34 female Sprague-Dawley rats (18 SAHs and 16 shams). Shams did not have the spinal needle descended to the.......

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The pre-chiasmatic single injection model of SAH mimics several important elements of human SAH, including the spike in ICP, reduction of CBF, transient global ischemia, upregulation of neuroinflammatory markers, and CVS14,15,16,18,19,20. The ICP-probe was also used as a port for intrathecal administration (

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The work was supported by the Lundbeck Foundation and the Lundbeck Grant of Excellence (no. R59-A5404). Funders had no role in any part of the manuscript.

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Name Company Catalog Number Comments
16 G peripheral vein catheter BD Venflon 393229 Needle shortened, distal 1 cm curved. Wings removed
Anesthesia bell/ chamber Unknown
Blood gas analyzer Radiometer ABL80
Blood pressure (BP) monitor Adinstruments ML117 Connects to Powerlab
Curved forceps, 12 cm x 3 F.S.T 11001-12 For anesthesia
Cylindrical pillow, 28 cm x 4 cm Homemade Made from surgical towels
Data acquisition hardware Adinstruments ML870 Powerlab
Data acquistion software Adinstruments LabChart 6.0
Drill KMD 1189
Drill controller Silfradent 300 IN
Flexible light Schott KL200
Heating pad Minco 1135
Hypodermic needle, 20 G KD Medical 301300 Connects to stereotaxic frame
ICP monitor Adinstruments ML117 Connects to Powerlab
Isoflurane vaporizer Ohmeda TEC3
Laptop Lenovo T410
Laser doppler monitor Adinstruments ML191
Laser doppler probe Oxford Optronics MSF100XP Connects to laser doppler monitor
Needle holder, 13 cm F.S.T 12001-13 For anesthesia
Precision syringe, 0.025 mL Hamilton 547407
Stereotaxic frame Kopf Instruments M900
Surgical microscope Carl Zeiss F170
Suture needle Allgaier 1245 For anesthesia
Temperaure controller CWE,INC. TC-1000
Transducer x 2 Adinstruments MLT0699 Connects to BP and ICP monitor
Ventilator Ugo Basile 7025
Veterinary clipper Aesculap GT421
3-pronged Blair retractor, 13.5 cm Agnthos 17022--13
Blunt Alm retractor F.S.T 17008-07
Curved forceps, 12 cm x 2 F.S.T 11001-12
Needle holder, 13 cm F.S.T 12001-13
Straight Dumont forceps, 11 cm F.S.T 11252-00
Straight Halsted-Mosquito hemostat x 2 F.S.T 13008-12
Straight Iris scissor, 9 cm F.S.T 14090-09
Straight Vannas scissor, 10.5 cm F.S.T 15018-10
Absorpable swabs Kettenbach 31603
Black silk thread, 4-0, 5 x 15 cm Vömel 14757
Bone wax Aesculap 1029754
Carbomer eye gel 2 mg/g Paranova
Cotton swab Heinz Herenz WA-1
Cotton tipped applicator x 4 Selefa 120788
Hypodermic needle, 23 G x2 KD Medical 900284 Connects to stopcock. Remove distal end
Hypodermic needle, 23 G x3 KD Medical 900284 Remove distal end. 2 connects to stopcock, 1 to syringe
ICP probe: Homemade Made of the following:
Polythene tubing, 20 mm Smiths medical 800/100/200 Inner diameter (ID): 0.58 mm, Outer diameter (OD): 0.96 mm.
Silicone tubing, 10 mm Fisher 15202710 ID: 0.76 mm, OD: 2.4 mm.
Silicone tubing, 2 mm Fisher 11716513 ID: 1.0 mm, OD: 3.0 mm.
Micro hematocrit tubes Brand 7493 11
OP-towel, 45 cm x75 cm Mölnlycke 800430
PinPort adapter, 22 G Instech PNP3F22
PinPort injector Instech PNP3M
Polythene tubing, 2 x 20 cm Smiths medical 800/100/200 Connects to syringe. ID: 0.58 mm, OD: 0.96 mm.
Rubberband Unknown
Scalpel, 10 blade Kiato 23110
Spinalneedle, 25 G x 3.5'' Braun 5405905-01
Stopcock system, Discofix x 2 Braun 16494C Connects to transducer
Suture, 4-0, monofil, non-resorbable x 3 Ethicon EH7145H
Syringe, 1 mL BD Plastipak 1710023
Syringe, luer-lock, 10 mL x 4 BD Plastipak 305959 Connects to transducer
Tissue adhesive glue 3M 1469SB
0.5% Chlorhexidine spirit Faaborg Pharma 210918
Carprofen 50 mg/mL ScanVet 43715 Diluted 1:10
Isoflurane Baxter
Isotonic saline Amgros 16404
Lidocaine-Adrenaline 10 mg/5 µg/mL Amgros 16318

  1. van Gijn, J., Kerr, R. S., Rinkel, G. J. Subarachnoid haemorrhage. Lancet. 369 (9558), 306-318 (2007).
  2. de Rooij, N. K., Linn, F. H. H., vander Plas, J. A., Algra, A., Rinkel, G. J. E. Incidence of subarachnoid haemorrhage: a systematic review with emphasis on region, age, gender and time trends. Journal of Neurology, Neurosurgery, and Psychiatry. 78 (12), 1365-1372 (2007).
  3. Feigin, V. L., Lawes, C. M., Bennett, D. A., Barker-Collo, S. L., Parag, V. Worldwide stroke incidence and early case fatality reported in 56 population-based studies: a systematic review. The Lancet, Neurology. 8 (4), 355-369 (2009).
  4. Maher, M., Schweizer, T. A., Macdonald, R. L. Treatment of spontaneous subarachnoid hemorrhage: guidelines and gaps. Stroke. 51 (4), 1326-1332 (2020).
  5. Pickard, J. D., et al. Effect of oral nimodipine on cerebral infarction and outcome after subarachnoid haemorrhage: British aneurysm nimodipine trial. British Medical Journal (Clinical Research ed.). 298 (6674), 636-642 (1989).
  6. Daou, B. J., Koduri, S., Thompson, B. G., Chaudhary, N., Pandey, A. S. Clinical and experimental aspects of aneurysmal subarachnoid hemorrhage. CNS Neuroscience and Therapeutics. 25 (10), 1096-1112 (2019).
  7. Fujii, M., et al. Early brain injury, an evolving frontier in subarachnoid hemorrhage research. Translational Stroke Research. 4 (4), 432-446 (2013).
  8. Roos, Y. B., et al. Complications and outcome in patients with aneurysmal subarachnoid haemorrhage: A prospective hospital based cohort study in the Netherlands. Journal of Neurology, Neurosurgery, and Psychiatry. 68 (3), 337-341 (2000).
  9. Vergouwen, M. D. I., et al. Definition of delayed cerebral ischemia after aneurysmal subarachnoid hemorrhage as an outcome event in clinical trials and observational studies: proposal of a multidisciplinary research group. Stroke. 41 (10), 2391-2395 (2010).
  10. Brown, R. J., Kumar, A., Dhar, R., Sampson, T. R., Diringer, M. N. The relationship between delayed infarcts and angiographic vasospasm after aneurysmal subarachnoid hemorrhage. Neurosurgery. 72 (5), 702-707 (2013).
  11. Dhar, R., et al. Relationship between angiographic vasospasm and regional hypoperfusion in aneurysmal subarachnoid hemorrhage. Stroke. 43 (7), 1788-1794 (2012).
  12. Macdonald, R. L. Delayed neurological deterioration after subarachnoid haemorrhage. Nature Reviews. Neurology. 10 (1), 44-58 (2014).
  13. Marbacher, S., et al. Systematic review of in vivo animal models of subarachnoid hemorrhage: species, standard parameters, and outcomes. Translational Stroke Research. 10 (3), 250-258 (2019).
  14. Prunell, G. F., Mathiesen, T., Svendgaard, N. -. A. A new experimental model in rats for study of the pathophysiology of subarachnoid hemorrhage. Neuroreport. 13 (18), 2553-2556 (2002).
  15. Prunell, G. F., Mathiesen, T., Diemer, N. H., Svendgaard, N. A. Experimental subarachnoid hemorrhage: subarachnoid blood volume, mortality rate, neuronal death, cerebral blood flow, and perfusion pressure in three different rat models. Neurosurgery. 52 (1), 165-176 (2003).
  16. Prunell, G. F., et al. Experimental Subarachnoid Hemorrhage: Cerebral blood flow and brain metabolism during the acute phase in three different models in the rat. Neurosurgery. 54 (2), 426-437 (2004).
  17. Velthuis, B. K., et al. Subarachnoid hemorrhage: Aneurysm detection and preoperative evaluation with CT angiography. Radiology. 208 (2), 423-430 (1998).
  18. Leclerc, J. L., et al. A comparison of pathophysiology in humans and rodent models of subarachnoid hemorrhage. Frontiers in Molecular Neuroscience. 11, 71 (2018).
  19. Prunell, G. F., Svendgaard, N. A., Alkass, K., Mathiesen, T. Inflammation in the brain after experimental subarachnoid hemorrhage. Neurosurgery. 56 (5), 1082-1091 (2005).
  20. Prunell, G. F., Svendgaard, N. A., Alkass, K., Mathiesen, T. Delayed cell death related to acute cerebral blood flow changes following subarachnoid hemorrhage in the rat brain. Journal of Neurosurgery. 102 (6), 1046-1054 (2005).
  21. Spray, S., Haanes, K. A., Edvinsson, L., Johansson, S. E. Subacute phase of subarachnoid haemorrhage in female rats: increased intracranial pressure, vascular changes and impaired sensorimotor function. Microvascular Research. 135, 104127 (2020).
  22. Ansar, S., Vikman, P., Nielsen, M., Edvinsson, L. Cerebrovascular ETB, 5-HT1B, and AT1 receptor upregulation correlates with reduction in regional CBF after subarachnoid hemorrhage. American Journal of Physiology - Heart and Circulatory Physiology. 293 (6), 3750-3758 (2007).
  23. Hansen-Schwartz, J., et al. Subarachnoid hemorrhage enhances endothelin receptor expression and function in rat cerebral arteries. Neurosurgery. 52 (5), 1188-1194 (2003).
  24. Hayman, E. G., Wessell, A., Gerzanich, V., Sheth, K. N., Simard, J. M. Mechanisms of global cerebral edema formation in aneurysmal subarachnoid hemorrhage. Neurocritical Care. 26 (2), 301-310 (2017).
  25. Miyata, H., et al. Vasa vasorum formation is associated with rupture of intracranial aneurysms. Journal of Neurosurgery. , 1-11 (2019).
  26. Tada, Y., et al. Roles of hypertension in the rupture of intracranial aneurysms. Stroke. 45 (2), 579-586 (2014).
  27. Nuki, Y., et al. Elastase-induced intracranial aneurysms in hypertensive mice. Hypertension. 54 (6), 1337-1344 (1979).
  28. Marbacher, S., Wanderer, S., Strange, F., Grüter, B. E., Fandino, J. Saccular aneurysm models featuring growth and rupture: A systematic review. Brain Sciences. 10 (2), 101 (2020).
  29. Altay, O., et al. Isoflurane on brain inflammation. Neurobiology of Disease. 62, 365-371 (2014).
  30. Hockel, K., Trabold, R., Schöller, K., Török, E., Plesnila, N. Impact of anesthesia on pathophysiology and mortality following subarachnoid hemorrhage in rats. Experimental and Translational Stroke Medicine. 4 (1), 5 (2012).
  31. Kamp, M. A., et al. A Systematic and meta-analysis of mortality in experimental mouse models analyzing delayed cerebral ischemia after subarachnoid hemorrhage. Translational Stroke Research. 8 (3), 206-219 (2017).
  32. Povlsen, G. K., Johansson, S. E., Larsen, C. C., Samraj, A. K., Edvinsson, L. Early events triggering delayed vasoconstrictor receptor upregulation and cerebral ischemia after subarachnoid hemorrhage. BMC Neuroscience. 14, 34 (2013).

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