Characterization of tissue and functional deficits in a clinically translational pig model of acute ischemic stroke

Abstract

The acute stroke phase is a critical time frame used to evaluate stroke severity, therapeutic options, and prognosis while also serving as a major tool for the development of diagnostics. To further understand stroke pathophysiology and to enhance the development of treatments, our group developed a translational pig ischemic stroke model. In this study, the evolution of acute ischemic tissue damage, immune responses, and functional deficits were further characterized. Stroke was induced by middle cerebral artery occlusion in Landrace pigs. At 24 h post-stroke, magnetic resonance imaging revealed a decrease in ipsilateral diffusivity, an increase in hemispheric swelling resulting in notable midline shift, and intracerebral hemorrhage. Stroke negatively impacted white matter integrity with decreased fractional anisotropy values in the internal capsule. Like patients, pigs showed a reduction in circulating lymphocytes and a surge in neutrophils and band cells. Functional responses corresponded with structural changes through reductions in open field exploration and impairments in spatiotemporal gait parameters. Characterization of acute ischemic stroke in pigs provided important insights into tissue and functional-level assessments that could be used to identify potential biomarkers and improve preclinical testing of novel therapeutics.

Keywords: Acute stroke; Brain ischemia; Gait analysis; Magnetic resonance imaging; Porcine.

Figure 1:. MCAO induced acute ischemic infarction and decreased diffusivity.

DWI sequences exhibited territorial hyperintense lesions of 9.91±1.40 cm³ characteristic of an edematous injury (A, white arrow). ADC maps revealed signal void indicative of restricted diffusion and cytotoxic edema (B, white arrow). Ipsilateral ROIs exhibited a significantly (p≤0.0001) lower ADC value relative to the contralateral hemisphere (0.34±0.02 vs. 0.62±0.03 x10⁻³mm²/s, respectively; C). * indicates significant difference between hemispheres.

Figure 2:. Ischemic stroke resulted in hemispheric swelling, consequent MLS, and ICH.

T2W sequences revealed increased (p<0.01) swelling of the ipsilateral hemisphere (25.99±1.78 vs. 22.49±1.40 cm³; A-C) resulting in a pronounced MLS of 2.48±0.55 mm compared to pre-stroke imaging (A and B, red lines). Characteristic hypointense ROIs indicated the presence of ipsilateral ICH when compared to pre-stroke T2* sequences (1.73±0.17 cm³, D and E, white arrow).

Figure 3:. Ischemic stroke diminished WM integrity of the IC.

Pre-stroke the left and right IC possess similar WM integrity (A). 24 hours post-stroke, the ipsilateral IC exhibited a disruption in WM integrity (B, white arrow). Further analysis revealed a significant (p<0.01) decrease in the ipsilateral IC FA value when compared to the contralateral IC (0.17±0.01 vs. 0.23±0.01 respectively; C). * indicates significant difference between hemispheres.

Figure 4:. Ischemic stroke led to increased circulating neutrophil levels and decreased circulating lymphocyte levels.

Band neutrophils showed a significant (p<0.05) increase 12 hours post-stroke when compared to pre-stroke (5.5±1.09% vs. 1.92±0.99% respectively; A, B). Circulating neutrophils were significantly (p<0.05) increased at 4- and 12-hours post-stroke relative to pre-stroke (43.6±5.00% and 48.9±5.01% vs. 26.5±4.57%, respectively; C, D). Circulating lymphocytes were significantly (p<0.05) decreased at 4, 12, and 24-hours post-stroke compared to pre-stroke (31.57±4.64%, 25.60±4.66%, and 26.54±4.65% vs. 44.83±4.26%; E, F). * indicates significant difference between pre-stroke and post-stroke time points.

Figure 5:. MCAO led to functional disabilities and behavioral abnormalities.

Ethovision XT tracking software was used during OF testing to automatically assess differences in perimeter sniffing (red lines) pre-stroke (A) and post-stroke (B). Exploratory perimeter sniffing frequencies were significantly (p<0.05) reduced at 48 hours post-stroke compared to pre-stroke observations (26.0±4.02 vs. 13.0±2.94, respectively; C), whereas distance traveled was not (p=0.77; 85.48±8.48 vs. 79.74±19.59 m). Black lines were indicative of non-perimeter sniffing and other non-exploratory behaviors (e.g. ambulation, laying down, escape behavior). * indicates a significant difference from pre-stroke.

Figure 6:. Ischemic stroke resulted in spatiotemporal gait deficits.

Velocity and cadence significantly (p< 0.01) decreased post-stroke (61.01±8.39 vs 162.9±12.73 cm/s and 61.01±5.91 vs 126.44±3.72 steps/min, respectively, A-B). The LF swing percent of cycle significantly (p<0.01) decreased compared to pre-stroke (30.70±2.12 vs 48.89±2.35%, respectively, C). A significant (p<0.01) decrease in LF stride length was observed post-stroke compared to pre-stroke (59.04±3.85 vs 76.72±4.60cm, respectively, D). LF cycle time significantly (p<0.01) increased relative to pre-stroke (1.02±.09 vs 0.48±0.013sec, respectively, E). The mean pressure exhibited by the LF significantly (p<0.01) decreased at post-stroke compared to pre-stroke (2.62±.03 vs 2.82±.03 arbitrary units (A.U.), respectively, F). * indicates significant difference between pre-stroke and post-stroke time points.