Light irradiation after the injection of photosensitive dyes is an approach to develop an experimental stroke model in laboratory animal models [10, 11]. The generation of reactive oxygen species by a photosensitive dye triggers clotting cascades, leading to ischemia-induced pathological changes in the targeted region, which is applied to the human counterpart observed in the clinical setting [18]. In this research, a green laser at the wavelength of 562 nm and 150 mW, using cold light optic fibers was selected to activate RB. This approach enables us to conduct irradiation easily at a significantly low price [42]. Also, four circular polarizers were used to control output beam intensity and successfully activate light-sensitive dye without heat generation and unwanted burn in the target zone. The molecules of a polarizing filter were aligned in the same direction. Vibrating molecules could absorb the light waves that have the same orientation as the filter, therefore they decrease the intensity and scattering of the light passing through these filters [22]. In this experiment, the temperature of target zone reached 37°C after 10 minutes. The time of radiation did not exceed more than 10 minutes and this time was used to PT-induced brain ischemia. Therefore, based on our experiment, it seems that the application of the polarized laser beam could be the safe and reliable method for induction of PT stroke model [43]. In our previous experiment, we evaluated the histopathological changes from 1 to 7 days after PT induction using morphometric and stereological methods [44]. Here, in the present study, we decided to investigate whether this technique is reproducible or not and also, we would like to extend the follow up the focal cerebra ischemia outcomes on cerebral tissue architecture, particularly cellular behavior and neurobehavioral performance for a prolonged period (28 days) following to PT. Despite prominent weight alteration, the existence of insignificant causalities indicates the safety of the current model in the induction of ischemic stroke. Histological examination showed the ischemic brain changes as early as 1 day, indicated by disseminated hyperemia after PT-induced brain ischemia protocol. The pattern of hyperemia turned into ecchymosis vasodilation at day 3 which further became pale by progression of time after 5 days, confirming necrotic changes. Quantitative stereological analysis revealed the reduction of the infarct cavity volume, reaching to minimum levels on day 28. Since the level of neurological deficits and recovery after ischemia is deeply linked to both the infarct size and neurovascular structure of the peri-infarct area [45], therefore, it is not surprising that largest functional deficits were observed 1 to 7 days post ischemic that reduced gradually over time and recovery initiated thereafter. Bright-field microscopic imaging showed bulk degeneration and sloughing of ischemic areas after PT-induced ischemia on day 7, leading to the generation of vacant space. In agreement with these findings, Hailong et al., demonstrated that PT-induced mice showed the largest functional deficits during days 2–4 post stroke, that gradually and spontaneously recovered over time and reached the minimum level on days 14 after stroke. Infarct volume enhanced from 5 h and reached the largest size one to two days post-ischemia and thereafter decreased gradually by time. They reported the close correlation of functional impairment with brain damage and cellular proliferation [46].
In contrast to ischemic volume changes, the number of active astrocytes was increased and exhibited in the periphery of lesion sites with elongated and spindle-like fibroblast appearance. These cells tended to form scar gliosis by time. Additionally, massive chromatolysis such as cell soma swelling, and round-form appearance with eccentric nuclei were seen in the periphery of irradiated areas, contributing to neural cell death and degeneration. This data showed that astrogliosis is the main host tissue response in the injured brain. These features were correlated with dynamic activity of astrocytes and characteristic hallmarks of pro-inflammatory responses after occurrence of ischemic changes [47]. In the present study, the number of BrdU labeled cells significantly increased around the infarct cavity on day 28 compared to RB and laser irradiation groups, indicating increased cell proliferation and migration at the periphery of ischemic areas. Theoretically, it has been demonstrated that new neurons are produced under certain pathological conditions such as brain ischemia to regenerate the damaged region to improve the neurological functions [48]. We also performed double NeuN+/Brdu+ staining to measure the rate of neurogenesis at the periphery of lesion sites. In this regard, we calculated the NeuN+/BrdU+ ratio. Despite enhanced proliferation rate at the periphery of lesion sites in the RB + Laser irradiation group, our data showed that a small fraction of proliferating cells differentiated to the mature neurons compared to the other groups. Consistent with current data, some previous studies indicated that endogenous neurogenesis occurred after stroke and other ischemic insults [46, 49]. Even, neuronal differentiation was not completely stopped in our study but this capacity diminished significantly compared to the health condition. Abati et al indicated that SVZ- derived NSCs migrate to the damaged cortex after an injury, where a subpopulation will generate reactive astrocytes [50]. Furthermore, Faiz et al reported that SVZ-derived NSCs migrate to the stroke lesion and rapidly differentiate and give rise to a subpopulation of reactive astrocytes, suggesting a role for the SVZ in post-injury gliosis [51]. The lack of sufficient compensatory neurogenesis and small number of Brdu+/NeuN + cells, are due to the undesirable microenvironment, storm release of cytokines, and reduction of appropriate growth factors that coincided with increased differentiation towards astrocyte lineage [52].