Using multiple approaches, the following findings were observed after PT-induced ischemia: 1) Histological and serial MRI data showed that brain infarction can be observed as early as 5 h after PT, and reached a peak on days 1–3 after PT before declining thereafter. Significant brain swelling and edema were observed in vivo in the same time frame after PT from MRI. 2) The Brdu labeled proliferating cells exhibit temporal and spatial changes after PT. The rate of Brdu+ cell generation is higher in the region close to ischemic core and reaches the highest level within post ischemic days 3 to 4. Similar time courses were observed for the generations of proliferating GFAP+ astrocytes and Iba1+ microglia/macrophages. 3) The percentage of GFAP+ Brdu+ reactive astrocytes out of total Brdu+ cells was small in each time interval, with the highest rate of ~6% within post ischemic days 5 to 6. 4) The largest sensory-motor functional deficits were observed on post ischemic days 2–4 with beginning recovery detected thereafter. Thus, the current study found that the first a few days is a critical period that causes brain expansion, cellular proliferation and behavioral deficits after PT and functional deficits are positively correlated with brain infarct expansion as well as swelling and edema, and cellular proliferation.
Our studies from histochemistry, and T2WIs and DWIs of MRI of live mice indicate that infarction expanded to a maximum 1–3 days after the onset of PT and gradually reduced over the time course of two weeks. Nissl staining showed clear glial scar formation around the border of ischemic region six days after PT. Based on ADC maps and T2WIs, we observed significant vasogenic and cytotoxic edema and brain swelling within the first three days after PT, and a significant decrease and atrophy after day 6. Data of a time-dependent decline in lesion and swelling after day 6 from MRI, together with Nissl staining, indicate that the expansion of infarction was accompanied by brain edema development. The spontaneous recovery of brain injury and glial scar formation begum from about day 6, consistent with other reports [21, 34]. Our results are consistent with clinical study using serial MRI on patients after stroke, which revealed an increase in stroke volume from several hours to 3–5 days after stroke onset  Interestingly, both T2WIs and DWIs can detect ischemic lesion as early as 5 h after PT when demarcation is not clear and penumbral ischemic tissue is salvageable using tPA in clinic, suggesting that MRI is a useful tool for the detection of very acute ischemia.
Astrocytes experience a variety of alterations in brain disorders . Astrocytes can exhibit enhanced Ca2+ signaling and are known to become reactive over time after ischemia as characterized by an excessive expression of GFAP (i.e., astrogliosis), and eventually form a glial scar surrounding the area of the ischemic core [5, 16, 29]. Reactive astrocytes could be formed from the transformation of existing astrocytes or newly generated astrocytes with a common feature of high GFAP expression levels [12, 16, 17, 20, 37]. Although spatial and temporal dependence of cellular proliferation was investigated [16, 19, 20], systematic and detailed dynamic studies on cellular proliferation and glial scar formation were lacking. We designed a ‘time-block’ Brdu injection protocol to precisely label the proliferating cells at different times after PT. Our results show that Brdu+ proliferating cells and GFAP+ Brdu+ proliferating reactive astrocytes were generated at the highest rate within post ischemic days 3 to 4 and then quickly reduced, and that proliferating astrocytes only accounted for small percentage of Brdu+ cells in each time period studied with the highest percentage being 5.8%. Thus our results demonstrated that the majority of GFAP+ reactive astrocytes resulted from the upregulation of GFAP in existing astrocytes without proliferation, and that the generation of proliferating astrocytes was highly time-dependent during post ischemia. The low proliferating rate of reactive astrocytes after a stroke is consistent with a report from Barret et al. although they used a different Brdu labeling protocol and ischemic model , in addition, despite the agreement of their results with ours, they did not conduct a detailed time course study.
On the other hand, the results of our study also revealed the persistence of a high GFAP+ cell density even in the distant region from ischemic core up to two weeks after PT, although the rate of GFAP+ Brdu+ cell generation was dramatically reduced after day 6. Glial scar is clearly formed from day 6 after ischemia. The GFAP+ reactive astrocytes experienced significant changes in morphology characterized as hypertrophy and stellate before day 6, as they became densely packed and formed a stream with straight and elongated processes pointing towards the ischemic core after day 6, thereby sharply separating the ischemic core area from unaffected issue.
Besides astrocytes, microglia also played a crucial role in cellular proliferation and glial scar formation post stroke. As a major source of proinflammatory factors in brain, microglia is significantly activated after ischemic stroke [18, 19]. Our results showed that the dynamic changes of Iba1+ cells and Iba1+ Brdu+ cells followed the same pattern as GFAP+ and GFAP+ Brdu+ cells, i.e., with the highest rates recorded within post ischemic days 3 to 4. Nevertheless, a large increase in Iba1+ Brdu+ microglia also took place within post ischemic days 1 to 2, and the response receded more quickly compared with GFAP+ reactive astrocytes with undetectable proliferating Iba1+ microglia from day 10 after ischemia. Thus, compared with astrocytes, Iba1+ microglia recruits to ischemic regions more rapidly in response to ischemia. Another difference between astrocytes and microglia is that the percentage of proliferating microglia from total proliferating cells is much higher than the percentage of proliferating reactive astrocytes after PT. This is consistent with a recent study showing that the resident microglia, not infiltrated macrophages, contribute to proliferating microglia after PT . Microglia has been described to play both beneficial and detrimental roles in stroke . The rapid recruitment of microglia is consistent with the observation that microglia are the first barrier of the central nervous system, respond immediately to exogenous stimulation through increased inflammatory signals thereby contributing to the destruction of the infectious agents to protect sensitive neural tissue by removing cell debris and engulfing invading neutrophil granuocytes [38, 39]. Together with the same time course of lesion expansion and edema and swelling, our data suggest that the first few days after ischemia is a critical time window for microglia to exert beneficial or detrimental effects after ischemia. In addition, the percentage of proliferating Iba1+ microglia in total Brdu+ cells was much larger than that of proliferating GFAP+ cells. Since Iba1 is also expressed in macrophages [20, 40], the infiltration of macrophage might contribute greatly to total Iba1+ cells after ischemia, but on the other hand, this also suggests that stroke-induced increase of proliferated microglia might play a role in maintaining their continuous surveillance function and supporting glial scar formation. After day 6, densely packed Iba1+ cells in the ischemic core region were observed concomitant with astrocyte-derived scar formation, suggesting that both astrocytes and microglia contribute to scar formation.
Based on our Brdu+ labeling protocol, no mature neurons (Brdu+/NeuN+) were found in the penumbra. This is probably because the Brdu labeling time (i.e., two days between Brdu+ injection and transcardial perfusion in our protocol) is too short to form mature neurons in the penumbra in the cortex. However, mature neurons can be found in the cortex under normal and ischemic conditions using protocols with multiple daily injections of Brdu and prolonged labeling and waiting periods before immunocytochemical study [41–43]. On the other hand, although we did not examine the time course of apoptotic neuronal death, it is time dependent from our and other study based on TUNEL and caspase-3 staining [12, 20].
After ischemia, the brain will experience spontaneous recovery process [3, 31, 44]. However, it is still poorly understood how infarct expansion, cellular proliferation and reactive gliosis are correlated with functional deficits. Our data from behavioral study showed that PT significantly affected forelimb shift asymmetricity, strength, and sensory motor impairments in post ischemic days 2–4, which is in a similar time window with the infarct expansion, brain edema and swelling formation, and the highest rates of cell proliferation and reactive astrocyte generation. It is still controversial whether glial scar has beneficial or detrimental effects on neuronal and brain protection after ischemia. Attenuation of glial scar formation has been associated with both increases and decreases in infarct volumes and functional recovery [12, 15, 21, 37]. Double knockout mice of GFAP and vimentin have larger infarction than WT mice after ischemia . The current study shows that functional deficit is recovered from day 6 after ischemia when glial scar starts to form, suggesting that glial scarring might have a beneficial effect by stopping the expansion of the ischemic core. Although there have been reports regarding behavioral recovery after photothrombotic ischemia in mice, it appears that these behavioral tests have only been conducted after day 7 of post ischemia or were only conducted for a single post ischemic time point in past studies [3, 4, 46], and consequently missed critical information in the acute phase and during recovery process. Our study revealed that brain damage including infarct expansion and brain swelling as well as edema, and cellular proliferation are positively correlated with behavioral deficits in the acute phase. Behavioral recovery starts 6 days after PT when infarct expansion, brain edema and swelling, and cellular proliferation are decreased.