Changes in cellular signaling patterns before and after stroke in the middle cerebral arteries of stroke prone spontaneously hypertensive rats

Background Hemorrhagic stroke is associated with loss of middle cerebral artery (MCA) autoregulation in the stroke-prone spontaneously hypertensive rat (SHRsp). The signaling mechanism associated with the functional loss has yet to be defined. We hypothesize that physiological alterations coincide with changes to cerebrovascular inflammatory and contractile signaling and altered calcium signaling. METHODS: SHRsp rats were fed a high salt (4% NaCl) diet and sacrificed at 9 weeks of age for pre-stroke and after evidence of stroke for post-stroke samples. The MCAs were isolated for measuring protein levels using immunofluorescence (IF) & western blot (WB) for inflammatory signaling and contractile proteins. Tissues surrounding the MCA were analyzed for neuro-inflammation, neuronal damage, total and activated inflammatory proteins (ERK1/2 and p38MAPK), cerebrovascular contraction (PKC and MLC), and transient receptor potential V4 (TRPV4) expression. RESULTS: Our data show increase in activated inflammatory proteins after stroke with an associated decrease in expression of activated contractile proteins and TRPV4 channel expression compared to pre-stroke MCA. The post-stroke samples also show significant increase in neuro-inflammation and neuronal damage compared to pre-stroke samples. CONCLUSION An increase in activated/total (p38 MAPK &ERK1/2) is accompanied by a decrease in activated/total PKC & TRPV4 channel expression in post-stroke SHRsps. The decrease in vessel structural integrity and altered vascular tone of the MCAs may affect its ability to contract in response to pressure. Significant neuro-inflammation and neuronal damage in the brain tissues surrounding the MCA in post-stroke samples suggest MCA dysfunction is accompanied with neuronal and neural damage during stroke.

4 67 stroke (16-18) and may be involved in the underlying signalling changes taking place within the 68 MCA as stroke develops and PDC is lost. 69 Previous studies on isolated post-stroke MCAs have shown diminished response to PKC agonists, 70 as well as loss of NOS activity, indicating functional deterioration of both the endothelial and 71 vascular smooth muscle functions in the MCA(19). However, the underlying mechanism affecting 72 the functioning of the vessel during stroke has not been investigated. The current work helps to 73 identify the potential changes in protein expression occurring in the MCA before and after stroke 74 in the SHRsp animal model. The study also investigates the extent of neuro-inflammation and 75 neuronal damage in the brain tissue surrounding the MCA in pre-stroke and post-stroke SHRsps. 76 The determination of cellular signalling patterns in MCA will help understand the vascular 77 progression of stroke and its accompanying changes, leading to potential treatment options. immunofluorescence analysis, MCAs were cut alongside surrounding brain tissue, placed in a chip 113 with embedding medium (Tissue Tek; Sakura Finetech Inc., CA, USA) and, flash frozen using 114 liquid nitrogen prior to storage at -80 C. The remaining brain tissue was fixed in 10% neutral 115 buffered formalin and embedded in paraffin for histological examination of neurological damage.

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In order to map potential areas of inflammation, sections were cut of brain tissue adjacent to the 160 MCA and were stained for astrocytes and microglia/macrophages using GFAP-Cy3 (1:1,000;      276 There was no difference however for total ERK and total p38MPAK relative to GAPDH in either of 277 the test groups indicating no change in total protein expression (results not shown).  The activation of inflammatory signalling markers in the MCA suggested that the brain itself may 286 have been exposed to inflammatory damage as well. Therefore, microglia and astrocytes were 287 imaged using brain slices immuno-stained with DAPI (nuclear stain), Iba-1(for total microglia)   The brain slices from the pre-stroke and post-stroke SHRsp rats were stained using H&E staining 304 and imaged to determine degrees of intracerebral and neural damage, particularly in the cortex in 305 proximity to the middle cerebral artery (Figures 8A, B). The following four parameters were 306 evaluated to determine the extent of damage in the brain during stroke using a semi-quantifiable  MCAs would directly affect vascular ECM stability(54) and integrity.

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MAPK activity is also linked with microglial activation due to increased inflammatory activity.

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In striatum of rats that had induced ICH, inhibition of MAPK pathways decreased survival of 416 activated microglia (55) and phosphorylated p38 MAPK localizes to neurons and microglia of 417 ischemic brain tissue in the hippocampus (56). The brain regions surrounding the post-stroke 418 MCAs, the anterior region extending from insula with the opercular segments (parietal and 419 temporal), showed significantly higher levels of activated microglia compared to the brain regions have been associated with cell death, and are associated with a broad range of inductive stimuli 447 (66). Post-stroke brain regions near the MCA showed elevated levels of neural damage, relative 448 to pre-stroke, with degenerating neurons and cell vacuolation as the most prominent differences 449 observed, indicating substantial neural damage in the brain regions near the MCA following stroke. 450 Axon degeneration has been widely seen in neurodegenerative diseases, including stroke and 451 motor neuropathies (67), and is associated with impaired transmission across the neural network 452 and cytoskeletal breakdown (67). This would cause a significant decrease in the neural network, 453 making the brain regions following stroke less responsive and functional.

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Our results showed significant differences in inflammatory and contractile signalling pathways 455 before and after stroke, however the exact timeframe of damage is unknown. It is unclear if many 456 of the signalling changes we have identified occur just prior to, or follow from the consequences 457 of the stroke event and what other structural changes occur in the vessels to promote dysfunction. 458 We demonstrate that the combination of increased inflammatory expression and decreased