J Korean Neurosurg Soc.  2024 May;67(3):333-344. 10.3340/jkns.2023.0154.

Prospero Homeobox 1 and Doublecortin Correlate with Neural Damage after Ischemic Stroke

• Lee DH
• Lee EC
• Park SW
• Lee Jy
• Kim KP
• Oh JS

• KMID: 2554871
• DOI: http://doi.org/10.3340/jkns.2023.0154

Abstract

Objective
: Markers of neuroinflammation during ischemic stroke are well characterized, but additional markers of neural damage are lacking. The study identified associations of behavioral disorders after stroke with histologic neural damage and molecular biological change.
Methods
: Eight-week-old, 25 g male mice of the C57BL/6J strain were subjected to middle cerebral artery occlusion (MCAO) to induce ischemic stroke. The control group was a healthy wild type (WT), and the experimental group were designed as a low severity MCAO1 and a high severity MCAO2 based on post-stroke neurological scoring. All groups underwent behavioral tests, realtime polymerase chain reaction, triphenyltetrazolium chloride (TTC) staining and Hematoxylin and Eosin staining. One-way analysis of variance was used to analyze statistical significance between groups.
Results
: In TTC staining, MCAO1 showed 29.02% and MCAO2 showed 38.94% infarct volume (p<0.0001). The pro-inflammatory cytokine interleukin (IL)-1β was most highly expressed in MCAO2 (WT 0.44 vs. MCAO1 2.69 vs. MCAO2 5.02, p<0.0001). From the distance to target in the Barnes maze test, WT had a distance of 178 cm, MCAO1 had a distance of 276 cm, and MCAO2 had a distance of 1051 (p=0.0015). The latency to target was 13.3 seconds for WT, 27.9 seconds for MCAO1, and 87.9 seconds for MCAO2 (p=0.0007). Prospero homeobox 1 (Prox1) was most highly expressed in MCAO2 (p=0.0004). Doublecortin (Dcx) was most highly expressed in MCAO2 (p<0.0001).
Conclusion
: The study demonstrated that histological damage to neural cells and changes in brain mRNA expression were associated with behavioral impairment after ischemic stroke. Prox1 and Dcx may be biomarkers of neural damage associated with long-term cognitive decline, and increased expression at the mRNA level was consistent with neural damage and long-term cognitive dysfunction.

Keyword

Ischemic stroke; Cerebral infarction; Middle cerebral artery occlusion; Long-term memory

Figure

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  Fig. 1. A : Overview of experimental design in the MCAO mouse model. All MCAO mice are neurologically scored post-operatively and undergo behavioral experiments sequentially from low to high stress starting on day 7. Upon completion of the behavioral experiments, brain tissue is harvested by euthanasia and sampled for mRNA level expression and histological staining. B : MCAO surgery to induce ischemic stroke in mice. blood flow to the CCA, ICA, and ECA is blocked via a vascular clamp, and then an MCAO suture is inserted through an incision in the ECA to achieve M1 site occlusion. C : Advancing a filament through an ECA incision in a mouse to the MCA. Immediately before middle cerebral artery occlusion (x-axis of the graph : -1), blood flow in the middle cerebral artery averaged 400 BPU, and during occlusion, it was 0 BPU. After reperfusion, blood flow ranged from 300 BPU to 400 BPU, depending on the individual, confirming that reperfusion was achieved. MCAO : middle cerebral artery occlusion, NS : neurological score, OFT : open field test, ART : adhesive removal test, RT-PCR : real-time polymerase chain reaction, TTC : triphenyltetrazolium chloride, H&E : Hematoxylin and Eosin, ACA : anterior cerebral artery, MCA : middle cerebral arte, PCA : posterior cerebral artery, BA : basilar artery, ICA : internal carotid artery, PPA : pterygopalatine artery, ECA : external carotid artery, CCA : common carotid artery, BPU : blood perfusion units.

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  Fig. 2. A : Measurement of brain infarct volume and neurological scores in the MCAO group. TTc staining in WT, MCAO1, and MCAO2 groups. Measurement of infarct volume in MCAO1 and MCAO2. B : Measurement of neurological scores after MCAO. C : Locomotor activity and sensorimotor changes in the MCAO group. Measured the total distance traveled in the open field test and the adhesive removal latency of ART for the WT and MCAO groups. D : Neuroinflammation-related mRNA levels in the MCAO group. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. WT : wild type, MCAO : middle cerebral artery occlusion, NS : neurological score, OFT : open field test, ART : adhesive removal test, Vcam1 : vascular cell adhesion molecule 1, Icam1 : intercellular adhesion molecule 1, IL : interleukin, Mcp1 : monocyte chemoattractant protein 1, ccr2 : c-c chemokine receptor 2, TTC : triphenyltetrazolium chloride.

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  Fig. 3. Measuring positive long-term cognitive memory in the MCAO group. ***p<0.001. WT : wild type, MCAO : middle cerebral artery occlusion.

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  Fig. 4. Histological finding in hippocampus and neural biomarker mRNA levels in the MCAO group. A : Hematoxylin and Eosin (H&E) staining of two groups of WT and MCAO. Microscopy showing cellular arrangement and apoptosis in the hippocampal region with H&E staining (×350). B : mRNA levels of Prox1 and dcx in WT and MCAO groups. ***p<0.001, ****p<0.0001. WT : wild type, MCAO : middle cerebral artery occlusion, Prox1 : prospero homeobox 1, dcx : doublecortin.

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  Fig. 5. Overview of experiments to identify behavioral, histological, and molecular biological changes induced by neural damage after ischemic stroke. created a mouse model that mimics human ischemic stroke by inducing MCAO through surgical intervention. The mice were then evaluated for NS and categorized into low or high symptom groups based on the severity of their symptoms. The groups were then assessed for the impact of ischemic stroke through behavioral and histological analysis. At the end of the analysis, conclusions were drawn about two genes, Prox1 and dcx. WT : wild type, MCAO : middle cerebral artery occlusion, NS : neurological score, Prox1 : prospero homeobox 1, dcx : doublecortin, PCR : polymerase chain reaction, TTC : triphenyltetrazolium chloride, H&E : Hematoxylin and Eosin.

Reference

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