It has recently been identified that after motor cortex stroke, the ability of microglia processes to respond to local damage cues is lost from the thalamus, a major site of secondary neurodegeneration (SND). In this study, we combine a photothrombotic stroke model in mice, acute slice and fluorescent imaging to analyse the loss of microglia process responsiveness. The peri-infarct territories and thalamic areas of SND were investigated at time-points 3, 7, 14, 28 and 56 days after stroke. We confirmed the highly specific nature of non-responsive microglia processes to sites of SND. Non-responsiveness was at no time observed at the peri-infarct but started in the thalamus seven days post-stroke and persisted for 56 days. Loss of directed process extension is not a reflection of general functional paralysis as phagocytic function continued to increase over time. Additionally, we identified that somal PY was present on non-responsive microglia in the first two weeks after stroke but not at later time points. Finally, both classical microglia activation and loss of process extension are highly correlated with neuronal damage. Our findings highlight the importance of microglia, specifically microglia dynamic functions, to the progression of SND post-stroke, and their potential relevance as modulators or therapeutic targets during stroke recovery.
Stroke induces tissue death both at the site of infarction and at secondary sites connected to the primary infarction. This latter process has been referred to as secondary neurodegeneration (SND). Using predominantly fixed tissue analyses, microglia have been implicated in regulating the initial response at both damage sites post-stroke. In this study, we used acute slice based multiphoton imaging, to investigate microglia dynamic process movement in mice 14 days after a photothrombotic stroke. We evaluated the baseline motility and process responses to locally induced laser damage in both the peri-infarct (PI) territory and the ipsilateral thalamus, a major site of post-stroke SND. Our findings show that microglia process extension toward laser damage within the thalamus is lost, yet remains robustly intact within the PI territory. However, microglia at both sites displayed an activated morphology and elevated levels of commonly used activation markers (CD68, CD11b), indicating that the standardly used fixed tissue metrics of microglial "activity" are not necessarily predictive of microglia function. Analysis of the purinergic P Y receptor, a key regulator of microglia process extension, revealed an increased somal localization on nonresponsive microglia in the thalamus. To our knowledge, this is the first study to identify a non-responsive microglia phenotype specific to areas of SND post-stroke, which cannot be identified by the classical assessment of microglia activation but rather the localization of P Y to the soma.
In the current study, we were interested in investigating whether Low oxygen post-conditioning (LOPC) was capable of limiting the severity of stroke-induced secondary neurodegeneration (SND). To investigate the effect of LOPC we exposed adult male C57/BL6 mice to photothrombotic occlusion (PTO) of the motor and somatosensory cortex. This is known to induce progressive neurodegeneration in the thalamus within two weeks of infarction. Two days after PTO induction mice were randomly assigned to one of four groups: (i) LOPC-15 day exposure group; (ii) a LOPC 15 day exposure followed by a 15 day exposure to normal atmosphere; (iii) normal atmosphere for 15 days and (iv) normal atmosphere for 30 days (n = 20/group). We observed that LOPC reduced the extent of neuronal loss, as indicated by assessment of both area of loss and NeuN+ cell counts, within the thalamus. Additionally, we identified that LOPC reduced microglial activity and decreased activity within the excitotoxic signalling pathway of the NMDAR axis. Together, these findings suggest that LOPC limits neuronal death caused by excitotoxicity in sites of secondary damage and promotes neuronal survival. In conclusion, this work supports the potential of utilising LOPC to intervene in the sub-acute phase post-stroke to restrict the severity of SND.
Automated cell nucleus segmentation is the key to gain further insight into cell features and functionality which support computer-aided pathology in early diagnosis of diseases such as breast cancer and brain tumour. Despite considerable advances in automated segmentation, it still remains a challenging task to split heavily clustered nuclei due to intensity variations caused by noise and uneven absorption of stains. To address this problem, we propose a novel method applicable to variety of histopathological images stained for different proteins, with high speed, accuracy and level of automation. Our algorithm is initiated by applying a new locally adaptive thresholding method on watershed regions. Followed by a new splitting technique based on multilevel thresholding and the watershed algorithm to separate clustered nuclei. Finalized by a model-based merging step to eliminate oversegmentation and a model-based correction step to improve segmentation results and eliminate small objects. We have applied our method to three image datasets: breast cancer stained for hematoxylin and eosin (H&E), Drosophila Kc167 cells stained for DNA to label nuclei, and mature neurons stained for NeuN. Evaluated results show our method outperforms the state-of-the-art methods in terms of accuracy, precision, F1-measure, and computational time.
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