2,3,5-Triphenyltetrazolium chloride (TTC) staining is a commonly used method to determine the volume of the cerebral infarction in experimental stroke models. The TTC staining protocol is considered to interfere with downstream analyses, and it is unclear whether TTC-stained brain samples can be used for biochemistry analyses. However, there is evidence indicating that, with proper optimization and handling, TTC-stained brains may remain viable for protein analyses. In the present study, we aimed to rigorously assess whether TTC can reliably be used for western blotting of various markers. In this study, brain samples obtained from C57BL/6 male mice were treated with TTC (TTC+) or left untreated (TTC-) at 1 week after photothrombotic occlusion or sham surgery. Brain regions were dissected into infarct, thalamus, and hippocampus, and proteins were extracted by using radioimmunoprecipitation assay buffer. Protein levels of apoptosis, autophagy, neuronal, glial, vascular, and neurodegenerative-related markers were analyzed by western blotting. Our results showed that TTC+ brains display similar relative changes in most of the markers compared with TTC- brains. In addition, we validated that these analyses can be performed in the infarct as well as other brain regions such as the thalamus and hippocampus. Our findings demonstrate that TTC+ brains are reliable for protein analyses using western blotting. Widespread adoption of this approach will be key to lowering the number of animals used while maximizing data.
Cognitive impairment is common after stroke, and disturbances in hippocampal function are often involved, even in remote non-hippocampal injuries. In terms of hippocampal function, growth hormone (GH) is known to affects plasticity and cognition. We aimed to investigate whether GH treatment after an experimental cortical stroke could enhance remote hippocampal plasticity and the hippocampal-dependent visual discrimination task. C57BL6 male mice were subjected to cortical photothrombotic stroke. Stroke mice were then treated with either saline or GH at 48 h after occlusion for 28 days. We assessed learning and memory using mouse touchscreen platform for the visual discrimination task. We also evaluated markers of neural progenitor cells, synaptic plasticity and cerebrovascular remodelling in the hippocampal formation. GH treatment significantly improved the performance on visual discrimination task after stroke. We observed a concomitant increased number of bromodeoxyuridine-positive cells in the dentate gyrus of the hippocampus. We also detected increased protein levels and density of doublecortin, a neuronal precursor cells marker, as well as glutamate receptor 1 (GLuR1), a synaptic marker. These findings provide further neurobiological evidence for how GH treatment could be used to promote hippocampal plasticity in a remote region from the initial cortical injury, and thus enhance cognitive recovery after stroke.
Motor impairment is the most common and widely recognised clinical outcome after stroke. Current clinical practice in stroke rehabilitation focuses mainly on physical therapy, with no pharmacological intervention approved to facilitate functional recovery. Several studies have documented positive effects of growth hormone (GH) on cognitive function after stroke, but surprisingly, the effects on motor function remain unclear. In this study, photothrombotic occlusion targeting the motor and sensory cortex was induced in adult male mice. Two days post-stroke, mice were administered with recombinant human GH or saline, continuing for 28 days, followed by evaluation of motor function. Three days after initiation of the treatment, bromodeoxyuridine was administered for subsequent assessment of cell proliferation. Known neurorestorative processes within the peri-infarct area were evaluated by histological and biochemical analyses at 30 days post-stroke. This study demonstrated that GH treatment improves motor function after stroke by 50%-60%, as assessed using the cylinder and grid walk tests. Furthermore, the observed functional improvements occurred in parallel with a reduction in brain tissue loss, as well as increased cell proliferation, neurogenesis, increased synaptic plasticity and angiogenesis within the peri-infarct area. These findings provide new evidence about the potential therapeutic effects of GH in stroke recovery.
There is emerging evidence suggesting that a cortical stroke can cause delayed and remote hippocampal dysregulation, leading to cognitive impairment. In this study, we aimed to investigate motor and cognitive outcomes after experimental stroke, and their association with secondary neurodegenerative processes. Specifically, we used a photothrombotic stroke model targeting the motor and somatosensory cortices of mice. Motor function was assessed using the cylinder and grid walk tasks. Changes in cognition were assessed using a mouse touchscreen platform. Neuronal loss, gliosis and amyloid-β accumulation were investigated in the peri-infarct and ipsilateral hippocampal regions at 7, 28 and 84 days post-stroke. Our findings showed persistent impairment in cognitive function post-stroke, whilst there was a modest spontaneous motor recovery over the investigated period of 84 days. In the peri-infarct region, we detected a reduction in neuronal loss and decreased neuroinflammation over time post-stroke, which potentially explains the spontaneous motor recovery. Conversely, we observed persistent neuronal loss together with concomitant increased neuroinflammation and amyloid-β accumulation in the hippocampus, which likely accounts for the persistent cognitive dysfunction. Our findings indicate that cortical stroke induces secondary neurodegenerative processes in the hippocampus, a region remote from the primary infarct, potentially contributing to the progression of post-stroke cognitive impairment.
White matter tract (WMT) degeneration has been reported to occur following a stroke, and it is associated with post-stroke functional disturbances. White matter pathology has been suggested to be an independent predictor of post-stroke recovery. However, the factors that influence WMT remodeling are poorly understood. Cortisol is a steroid hormone released in response to prolonged stress, and elevated levels of cortisol have been reported to interfere with brain recovery. The objective of this study was to investigate the influence of corticosterone (CORT; the rodent equivalent of cortisol) on WMT structure post-stroke. Photothrombotic stroke (or sham surgery) was induced in 8-week-old male C57BL/6 mice. At 72 h, mice were exposed to standard drinking water ± CORT (100 µg/mL). After two weeks of CORT administration, mice were euthanised and brain tissue collected for histological and biochemical analysis of WMT (particularly the corpus callosum and corticospinal tract). CORT administration was associated with increased tissue loss within the ipsilateral hemisphere, and modest and inconsistent WMT reorganization. Further, a structural and molecular analysis of the WMT components suggested that CORT exerted effects over axons and glial cells. Our findings highlight that CORT at stress-like levels can moderately influence the reorganization and microstructure of WMT post-stroke.
We have previously demonstrated that a cortical stroke causes persistent impairment of hippocampal-dependent cognitive tasks concomitant with secondary neurodegenerative processes such as amyloid-β accumulation in the hippocampus, a region remote from the primary infarct. Interestingly, there is emerging evidence suggesting that deposition of amyloid-β around cerebral vessels may lead to cerebrovascular structural changes, neurovascular dysfunction, and disruption of blood-brain barrier integrity. However, there is limited knowledge about the temporal changes of hippocampal cerebrovasculature after cortical stroke. In the current study, we aimed to characterise the spatiotemporal cerebrovascular changes after cortical stroke. This was done using the photothrombotic stroke model targeting the motor and somatosensory cortices of mice. Cerebrovascular morphology as well as the co-localisation of amyloid-β with vasculature and blood-brain barrier integrity were assessed in the cortex and hippocampal regions at 7, 28 and 84 days post-stroke. Our findings showed transient cerebrovascular remodelling in the peri-infarct area up to 28 days post-stroke. Importantly, the cerebrovascular changes were extended beyond the peri-infarct region to the ipsilateral hippocampus and were sustained out to 84 days post-stroke. When investigating vessel diameter, we showed a decrease at 84 days in the peri-infarct and CA1 regions that were exacerbated in vessels with amyloid-β deposition. Lastly, we showed sustained vascular leakage in the peri-infarct and ipsilateral hippocampus, indicative of a compromised blood-brain-barrier. Our findings indicate that hippocampal vasculature may represent an important therapeutic target to mitigate the progression of post-stroke cognitive impairment.
Cognitive impairment is a common and disruptive outcome for stroke survivors, which is recognized to be notoriously difficult to treat. Previously, we have shown that low oxygen post-conditioning (LOPC) improves motor function and limits secondary neuronal loss in the thalamus after experimental stroke. There is also emerging evidence that LOPC may improve cognitive function post-stroke. In the current study we aimed to explore how exposure to LOPC may improve cognition post-stroke. Experimental stroke was induced using photothrombotic occlusion in adult, male C57BL/6 mice. At 72 h post-stroke animals were randomly assigned to either normal atmospheric air or to one of two low oxygen (11% O2) exposure groups (either 8 or 24 h/day for 14 days). Cognition was assessed during the treatment phase using a touchscreen based paired-associate learning assessment. At the end of treatment (17 days post-stroke) mice were euthanized and tissue was collected for subsequent histology and biochemical analysis. LOPC (both 8 and 24 h) enhanced learning and memory in the 2nd week post-stroke when compared with stroke animals exposed to atmospheric air. Additionally we observed LOPC was associated with lower levels of neuronal loss, the restoration of several vascular deficits, as well as a reduction in the severity of the amyloid-beta (Aβ) burden. These findings provide further insight into the pro-cognitive benefits of LOPC.