Characterization of chronic stroke animal models

To generate a chronic stroke model of rat and mice, the intraluminal transient middle cerebral artery occlusion method was carried out in rats and mice (Fig 1A). Twenty four hours after reperfusion, TTC staining showed that both rat and mouse stroke models developed clear infarction (Non-stained area) in the ipsilateral striatum and dorsolateral cortex verifying proper occlusion of the Middle cerebral artery (Fig 1B) We also assessed the structural integrity of the ischemic brain over the same 28-day period using MRI. The hypointense areas in the ipsilateral hemisphere were evident at the acute phase of both rat and mouse models. In the chronic phase, rat MCAo showed an infarct cavity as shown by hypointense areas in the ipsilateral hemisphere (Fig 2A). However, In a mouse model of chronic stroke, MRI did not show any hypointense areas suggesting that the infarct cavity was not formed in a chronic stroke mouse model (Fig 2B). Sham-operated animals did not show any hypointense areas in the brain. Further, we evaluated the tissue integrity of chronic stroke animal models by Hematoxylin & Eosin and Cresyl-violet staining at 1 month after ischemia-reperfusion injury and compared with the histology of acute stroke i.e.,

3 days after reperfusion and sham-operated animals (Fig 2 C, D). Hematoxylin

& Eosin and Cresyl violet staining of coronal brain sections at the level of striatum grossly depicts the edematous ipsilateral hemisphere with the faintly stained ischemic area in acute stroke while atrophied ipsilateral hemisphere in chronic stroke. The tissue integrity of the chronic stroke rat brain was worse than a chronic stroke mouse brain. Furthermore, the presence of cells bearing pycnotic nuclei and vacuolated cytoplasm were readily present in the ischemic area of the acute stroke brain. In the chronic stroke brain, cells bearing non-neuronal morphology occupied the corresponding ischemic core region.

Fig 1. Induction of middle cerebral artery occlusion (MCAo) Rat and mouse model.

A. Transient cerebral ischemia was induced by inserting silicon-coated nylon suture (4-0 for rats and 6-0 for mice) via the right external carotid artery (ECA) through the internal carotid artery (ICA) to block middle cerebral artery (MCA).

B. Infarction resulted from the occlusion of MCA was evaluated by TTC staining 24 hours after reperfusion.

Fig 2. A general overview of a brain tissue structure of chronic stroke animal models. The changes in brain tissue integrity were monitored by MRI, H&E staining and cresyl violet staining from acute (day 3) to chronic phase of stroke (Day 30) in both rat and mouse model of stroke. MCA occlusion resulted in infarction of striatum and dorsolateral cortex of both rat and mouse brain in the acute phase of the stroke. In the chronic phase, rat MCAo resulted in the infarct cavity while chronic mouse MCAo resulted in atrophied ipsilateral brain hemisphere (A, B). Hematoxylin & Eosin and Cresyl violet staining showed significant loss of neural cells in acute stroke. Cells with Pycnotic nuclei and vacuolated cytoplasm are evident in ischemic core and penumbra regions. In the chronic phase, the infiltration of glial cells (non-neuronal morphology) is evident in ischemic core and penumbra regions of the ischemic brain (C, D).

Immunostaining against the neuronal marker, NeuN, and astroglial marker, GFAP showed large NeuN and GFAP non-reactive areas corresponding to ischemic region suggestive of on-going cell death mechanisms in acute stroke brain. In the chronic phase, there were sparsely distributed NeuN+ Neurons in the peri-infarct region while GFAP+ cells made a glial scar delineating ischemic and normal viable tissue. Iba1+ microglia/macrophages were readily observed in the ischemic region of acute stroke brain and persisted in the chronic phase of the stroke (Fig 3 A, B). In the chronic phase of ischemic stroke, there is the presence of growth inhibitory glial scar forming a fine border between ischemic core and normal viable brain tissue (4 A). This glial scar primarily consists of reactive astrocytes, activated macrophages/microglia, and extracellular matrix molecules, predominantly chondroitin sulfate proteoglycans (CSPGs). We found that, in the brain of chronic stroke brain, GFAP positive astrocytes became hypertrophied and elongated their processes from penumbra into the infarct core (4 B). These astrocytes strongly upregulated GFAP protein, a hallmark of astrogliosis responding to ischemic stroke. We were also able to observe that the pronounced change in astrocytic morphology and GFAP expression in reactive astrocytes was also accompanied by the upregulation of intermediate filament protein, Nestin. Double immune-histochemical studies showed that GFAP-positive reactive astrocytes in the glial scar expressed a high amount of extracellular matrix-like Neurocan and CSPGs, but did not overlap with ED1

positive activated microglia/macrophages. GFAP and ED1 positive cells along with extracellular matrix molecules neurocan and CSPGs formed a layer around the ischemic lesion, suggesting the presence of inhibitory glial scar in the chronic stroke rat brain. The glial scar is positioned in such a way that it forms a border around the injury site and acts as a neuroprotective barrier to evading inflammatory cells.

Fig. 3. Temporal changes in neural cells in acute (Day 3) and chronic stroke (Day 30). Immunostaining showed large NeuN and GFAP non-reactive areas corresponding to ischemic region suggestive of on-going cell death mechanisms in acute stroke brain. Iba1 immunostaining revealed activation of resident microglia and infiltration of macrophages into the ischemic brain in the acute phase of the stroke. In the chronic phase, there were sparsely distributed NeuN+

Neurons in the peri-infarct region while GFAP+ cells made a dense glial scar delineating ischemic and normal viable brain tissue. Iba1+

microglia/macrophages with activated morphology densely populated in the ischemic brain in the chronic phase of the stroke.

Fig. 4.Characterization of Glial Scar in chronic stroke. A. Low magnification image of a chronic stroke brain stained with GFAP & MAP2 and Region of interest for characterizing glial Scar. B. In the chronic phase of ischemic stroke, there is the presence of growth inhibitory glial scar forming a fine border between ischemic core and normal viable brain tissue. This glial scar primarily consists of reactive astrocytes (GFAP/Nestin+), activated macrophages/microglia (Iba1/ED1+) and extracellular matrix molecules, predominantly chondroitin sulfate proteoglycans (CSPGs). Note GFAP positive astrocytes became hypertrophied and elongated their processes from penumbra

into the infarct core.

Overall, the MCAo mice exhibited poor performance in behavioral tests during the first week after ischemia, but their performance improved spontaneously over the time. The MCAo animals consistently showed higher NSS score compared to the sham group through the testing period (Fig. 5A) In corner tests, normal or sham-operated mice displayed similar tendency in right and left turning behavior when the animals reached the corner. However, the MCAo animals showed the right-sided bias in turning back since the animals had the paresis in the left side (Fig. 5 B). The laterality index, the indicator of right turning preference, was significantly increased in MCAo animals (p<0.05).

Within the span of 28 days the right turning bias in the ischemic group was partially reduced owing to spontaneous improvement in their sensory-motor function on the left side of the body. The sham-operated animals turned both the directions with almost equal probability, over the entire period of behavioral testing. In the pole test before the surgery, when the mice were placed on the top of the pole facing their head upward, mice in both the sham and MCAo groups could turn their head completely vertically downward within baseline value of 1 second and reached the floor within 4 sec, when they were placed on the top of the pole facing their head upward.Ttotal, the time taken to reach the floor, was significantly increased after MCAo (Fig. 5C). Although the MCAo mice showed profound improvement in the motor function (bradykinesia) over the time, the

sham- operated animals always took lesser time to reach the floor over the testing span of 1 month. In the rotarod test, the MCAo mice remained on the rotarod for lesser time than did the sham-operated mice for the first week, but the difference became non-significant between the groups after a week (Fig. 5D)

Fig. 5. Behavior assessment in chronic stroke animal models. Behavior tests were performed on acute i.e, day 3 and chronic i.e, day 30 after the MCAo surgery. All data are presented as means±S.E. Both Rat and mouse MCAo animals showed severe behavioral deficits in neurological severity scores and corner test compared to sham operated animals in acute as well as chronic phase.

Rotarod and adhesive removal test were sensitive to detect behavioral deficits in chronic stroke rat models. Rotarod test was not sensitive to detect behavioral deficits in chronic phase of chronic stroke mouse model. The latency to reach the floor from the top of the pole was increased in the mouse MCAo animals compared to the sham group. (t-test, *p<0.05, ** p<0.01)