Special thanks to the Gidday lab for their work in developing the RHP protocol, as well as the Neuro-Models Facility (UTSW) for their assistance in the tMCAo surgeries. This work was supported by grants from the American Heart Association (AMS), The Haggerty Center for Brain Injury and Repair (UTSW; AMS), and The Spastic Paralysis Research Foundation of the Illinois-Eastern Iowa District of Kiwanis International (JMG).

NOTE: This protocol was approved by the Institutional Animal Care and Use Committee (IACUC) at UT Southwestern Medical Center which abides by the National Institutes for Health (NIH) policy for experimental animal usage.
1. Repetitive Hypoxic Preconditioning
<ol>
	<li>Custom design four flowmeters on gas regulators and attach to standard 15 L induction chambers with PVC tubes to allow compressed gas from the oxygen (O2) tanks to flow into the chambers via an inlet port. See Equipm...

The authors have nothing to disclose.

A single exposure to systemic hypoxia (i.e., 2 hr of 11% O2) in mice &#8220;transiently&#8221; protects the brain from tMCAo,29 meaning the epigenetic response to the hypoxic preconditioning challenge is short-lasting, and the baseline phenotype is restored within days. Repetitive presentations of the hypoxic preconditioning stimulus dramatically extend the duration of the neuroprotective phenotype.6 Many studies have shown that the frequency, magnitude, and duration of the repet...

As the leading cause of adult disability and the fourth leading cause of death, stroke is one of the most debilitating disease states facing the adult population of the United States.1 Animal models of stroke allow for experimental investigation of new methods of reducing ischemic injury and improving post-stroke recovery. One novel avenue for such translational research is preconditioning. Preconditioning is the intentional use of a non-damaging stimulus to reduce damage from a subsequent, and more severe, injury.2 Hypoxic preconditioning has been shown to produce pleiotropic changes in the brain that provide protection against stroke in both in vivo and in vitro studies.3 However, a single exposure to hypoxia only offers short-term neuroprotection, inducing less than 72 hr of tolerance against ischemia in adult mice.4 Even after four weeks of 14 hr daily exposures to hypobaric hypoxia, Lin et al. found that neuroprotection was only sustained for one week.5 Repetitive hypoxic preconditioning (RHP) is characterized by stochastic variations in frequency, duration, and intensity of hypoxic exposures. In contrast to a single preconditioning challenge, RHP induces a cerebroprotective phenotype that lasts up to eight weeks in mice.6 RHP reduced infarct volumes, blood-brain barrier (BBB) disruption, vascular inflammation, and leukocyte diapedesis for weeks after the final hypoxic exposure. RHP specifically reduced inflammation in the ischemic brain by reducing T cell, monocyte, and macrophage populations, while maintaining B cell populations in the ischemic hemisphere.7 In fact, RHP induced an immunosuppressive phenotype in mice prior to any CNS injury, including stroke. RHP-treated B cells isolated from RHP-treated healthy mice exhibited a unique anti-inflammatory phenotype, with a downregulation of both antigen presentation and antibody production. The overall reduction in pro-inflammatory adaptive immune mechanisms makes RHP an excellent methodology to induce endogenous immunosuppression for not only CNS-specific inflammatory diseases, but also systemic injury or disease models that include a pro-inflammatory pathology.
RHP reduces both infarct volume and BBB disruption following a transient middle cerebral artery occlusion (tMCAo). Animal models of stroke, such as the commonly used tMCAo, dramatically improve the understanding of the pathophysiology of stroke, as well as the design of more effective neurotherapeutics. First developed by Koizumi et al., in 1986,8 the tMCAo procedure is a widely used method of inducing stroke in rodents and one of the preferred methods for investigating inflammation following reperfusion. As the methods for tMCAo evolve, the more recent use of silicone-coated filaments further reduce the risk of subarachnoid hemorrhage compared to other models9,10 and improve reliability, though unfortunately tMCAo often produces a wide variation in infarct volumes.11-13 Most of these studies delineate infarction regions in coronal brain sections by staining with 2,3,5- triphenyltetrazolium chloride (TTC), considered a gold standard for infarct quantification because it is a simple and inexpensive way to produce vivid, replicable results. TTC serves as a substrate of dehydrogenases present in mitochondria. When brain slices are exposed to the TTC solution, TTC is selectively taken into living cells where its non-soluble reduction product, formazan, precipitates to a deep red color in viable mitochondria. Because of mitochondrial dysfunction in the ischemic tissue, this tissue remains white, allowing for differentiation of damaged and healthy tissue.14
RHP also reduces BBB disruption in the ischemic hemisphere.6 Therefore, the dual quantification of BBB integrity within the same brains as TTC-based infarct volume determinations15 would provide useful information about the full efficacy of endogenous protection, and potential causal relations between BBB disruption and infarction in untreated and treated animals. The influx of peripheral blood through a disrupted BBB, secondary to stroke, increases leukocyte populations, pro-inflammatory cytokines, oxidative stress, vasogenic edema, and hemorrhagic transformation in the ischemic hemisphere, ultimately increasing the rates of infection and mortality in patients with ischemic stroke.16,17 A common method of measuring BBB disruption in animal models is through quantification of Evans blue (EB) dye leak into the brain.15,18-21 EB selectively binds to serum albumin, a globular protein (MW=65 kDa) that does not cross the BBB in uninjured animals.22 Following ischemic stroke, EB infiltrates the brain, and fluoresces at 620 nm, allowing for measurement of optical density within the perfused injured parenchyma.22 The optical density is directly proportional to the permeability of the BBB when EB has been washed out of the post-mortem cortical vasculature by transcardiac perfusion. With the immediate processing of TTC-stained brains in animals with EB administration, both the infarct volume and BBB disruption can be effectively quantified. It should be noted, however, that neuronal injury and BBB disruption are not concomitant processes in the post-stroke brain,23,24 so the selection of time of sacrifice is an important consideration.
The protocol that follows details the RHP method, the tMCAo method for inducing a temporary arterial occlusion that models middle cerebral artery occlusions in human patients, and the dual histological methods for determining neural and vascular stroke injury endpoints. TTC measures cell death and cumulative tissue damage, allowing for the quantification of an overall infarct volume, while EB provides for the hemispheric quantification of BBB damage.

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quantification of neurovascular protection following repetitive hypoxic preconditioning and transient middle cerebral artery occlusion in mice

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week protocol for repetitive hypoxic preconditioning (RHP) induces long-term protection against central nervous system (CNS) injury in a mouse model of focal ischemic stroke. RHP consists of 9 stochastic exposures to hypoxia that vary in both duration (2 or 4 hr) and intensity (8% and 11% O2). RHP reduces infarct volumes, blood-brain barrier (BBB) disruption, and the post-stroke inflammatory response for weeks following the last exposure to hypoxia, suggesting a long-term induction of an endogenous CNS-protective phenotype. The methodology for the dual quantification of infarct volume and BBB disruption is effective in assessing neurovascular protection in mice with RHP or other putative neuroprotectants. Adult male Swiss Webster mice were preconditioned by RHP or duration-equivalent exposures to 21% O2 (i.e. room air). A 60 min transient middle cerebral artery occlusion (tMCAo) was induced 2 weeks following the last hypoxic exposure. Both the occlusion and reperfusion were confirmed by transcranial laser Doppler flowmetry. Twenty-two hr after reperfusion, Evans Blue (EB) was intravenously administered through a tail vein injection. 2 hr later, animals were sacrificed by isoflurane overdose and brain sections were stained with 2,3,5- triphenyltetrazolium chloride (TTC). Infarcts volumes were then quantified. Next, EB was extracted from the tissue over 48 hr to determine BBB disruption after tMCAo. In summary, RHP is a simple protocol that can be replicated, with minimal cost, to induce long-term endogenous neurovascular protection from stroke injury in mice, with the translational potential for other CNS-based and systemic pro-inflammatory disease states.

This study included 25 male Swiss Webster mice that were 10 weeks of age at the start of randomization into RHP (n = 10) or 21% O2 (n = 15) groups. Two weeks after the final RHP exposure, surgical procedures were performed, with groups blinded and counterbalanced between days. Following tMCAo, 1 mouse died during post-operative recovery and 1 mouse was excluded from the study because it did not meet the reperfusion CBF criterion. Both excluded mice were from the 21% O2 group. In accordance with ARRI...

Watch this Scientific Journal Video about Quantification of Neurovascular Protection Following Repetitive Hypoxic Preconditioning and Transient Middle Cerebral Artery Occlusion in Mice at JoVE.com

Quantification of Neurovascular Protection Following Repetitive Hypoxic Preconditioning and Transient Middle Cerebral Artery Occlusion in Mice

This protocol describes repetitive hypoxic preconditioning, or brief exposures to systemic hypoxia that reduce infarct volumes and blood-brain barrier disruption following transient middle cerebral artery occlusion in mice. It also details dual quantification of infarct volume and blood-brain barrier disruption after stroke to assess the efficacy of neurovascular protection.

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week ...

quantification-neurovascular-protection-following-repetitive-hypoxic

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JoVE Journal

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13.6K vistas.  University of Texas Southwestern Medical Center. This protocol describes repetitive hypoxic preconditioning, or brief exposures to systemic hypoxia that reduce infarct volumes and blood-brain barrier disruption following transient middle cerebral artery occlusion in mice. It also details dual quantification of infarct volume and blood-brain barrier disruption after stroke to assess the efficacy of neurovascular protection.