A Novel Improved Thromboembolism-Based Rat Stroke Model That Meets the Latest Standards in Preclinical Studies

Abstract

The animal thromboembolic model of ischemia perfectly mimics human ischemic stroke which remains the leading cause of disability and mortality in humans. The development of new treatment strategies was therefore imperative. The purpose of this study is to improve the thromboembolic stroke model in rats in order to design experiments that use motor tests, and are in accordance with the 3R principles to prevent complications and maintain the same size of the infarct repeatedly. Tail vein dye application, a protective skull mask and a stress minimization protocol were used as additional modifications to the animal stroke model. These modifications significantly minimized the pain and stress severity of the procedures in this model. In our experimental group of Long-Evans rats, a photo-induced stroke was caused by the application of a photosensitive dye (Rose Bengal) activated with white-light irradiation, thus eliminating the need to perform a craniotomy. The animals’ neurological status was evaluated using a runway elevated test. Histological examination of the brain tissue was performed at 12, 24 and 48 h, and seven days post-stroke. Tissue examination revealed necrotic foci in the cortex and the subcortical regions of the ipsilateral hemisphere in all experimental groups. Changes in the area, width and depth of the necrotic focus were observed over time. All the experimental groups showed motor disturbances after stroke survival. In the proposed model, photochemically-induced stroke caused long-term motor deficits, showed high reproducibility and low mortality rates. Consequently, the animals could participate in motor tests which are particularly suitable for assessing the efficacy of neuro-regenerative therapies, while remaining in line with the latest trends in animal experimental design.

After Ischemic Stroke, Minocycline Promotes a Protective Response in Neurons via the RNA-Binding Protein HuR, with a Positive Impact on Motor Performance

“…In the present study, we also assess whether the positive biochemical and morphological changes observed under the influence of minocycline would translate into improved motor functions. In this regard, we would like to emphasize that our improved and minimally invasive animal model of ischemic stroke allows for motor tests to be performed after FCI [17]. The data from selected tests showed positive changes in gait, balance level, speed of movement, and leg strength.…”

“…For this purpose, all the stroke animals underwent motor tests including the Horizontal Runway Elevated test (HRE), Grip Strength test (GST) and CatWalk™ XT. None of the animals had any health problems or complications following the procedures, and no animals were excluded from the tests, except for groups S1 and S1 + m, which had a short duration between general anesthesia and euthanasia, making it impractical to perform motor tests [17]. Using the GST test, we were able to analyze the strength of the front paws in stroke rats.…”

“…They had ad libitum access to water and standard rat chow. From the 5th week of the animals' life until the start of the study, routine behaviors were implemented according to our protocol (see our previous publication [17]), resulting in a significant stress reduction in the animals.…”

“…Within this context, the use of animal models is one of the most valid strategies to develop effective therapies [11,13]. Notably, the animal model of focal cerebral ischemia (FCI) allows for more reproducible results compared to other models [14][15][16] and closely mimics what happens in humans (i.e., the ischemic area takes a relatively small area of the entire cerebral cortex) [17,18]. Furthermore, this model is characterized by a low burden on the animals and enables the verification of the tested substance's effect through motor tests [17,19].…”

“…Notably, the animal model of focal cerebral ischemia (FCI) allows for more reproducible results compared to other models [14][15][16] and closely mimics what happens in humans (i.e., the ischemic area takes a relatively small area of the entire cerebral cortex) [17,18]. Furthermore, this model is characterized by a low burden on the animals and enables the verification of the tested substance's effect through motor tests [17,19]. A poor prognosis in ischemic stroke results from the irreversible loss of neurons [20], which is due to an abrupt blood flow shortage and excitotoxicity.…”