Assessing the Effects of Cytoprotectants on Selective Neuronal Loss, Sensorimotor Deficit and Microglial Activation after Temporary Middle Cerebral Occlusion

Abstract

Although early reperfusion after stroke salvages the still-viable ischemic tissue, peri-infarct selective neuronal loss (SNL) can cause sensorimotor deficits (SMD). We designed a longitudinal protocol to assess the effects of cytoprotectants on SMD, microglial activation (MA) and SNL, and specifically tested whether the KCa3.1-blocker TRAM-34 would prevent SNL. Spontaneously hypertensive rats underwent 15 min middle-cerebral artery occlusion and were randomized into control or treatment group, which received TRAM-34 intraperitoneally for 4 weeks starting 12 h after reperfusion. SMD was assessed longitudinally using the sticky-label test. MA was quantified at day 14 using in vivo [11C]-PK111195 positron emission tomography (PET), and again across the same regions-of-interest template by immunofluorescence together with SNL at day 28. SMD recovered significantly faster in the treated group (p = 0.004). On PET, MA was present in 5/6 rats in each group, with no significant between-group difference. On immunofluorescence, both SNL and MA were present in 5/6 control rats and 4/6 TRAM-34 rats, with a non-significantly lower degree of MA but a significantly (p = 0.009) lower degree of SNL in the treated group. These findings document the utility of our longitudinal protocol and suggest that TRAM-34 reduces SNL and hastens behavioural recovery without marked MA blocking at the assessed time-points.

Neuronal Loss after Stroke Due to Microglial Phagocytosis of Stressed Neurons

“…This is consistent with (but does not show) microglial activation, causing delayed neuronal loss. However, an inhibitor of microglial activation (TRAM-34, a blocker of calcium-activated potassium channels) prevented the delayed neuronal loss and behavioral deficits induced by 15 min MCAO in hypertensive rats [12]. This suggests that microglial activation caused delayed neuronal loss.…”

“…Stoke rapidly induces necrosis of all cells within the infarct; however, two types/locations of delayed neuronal death after brain ischemia have also been identified (Figure 1). ( 1) 'Selective neuronal loss' may occur in peri-infact areas 1-7 days after transient middle cerebral artery occlusion (MCAO) in rodent striatum (and to a lesser extent in the cortex), but may increase over the following weeks [4,[7][8][9][10][11][12][13]. (2) 'Secondary neurodegeneration' of the thalamus may occur 0.5-12 months after cortical stroke [2,3,14].…”

“…Some published examples of delayed neuronal death are presented in Table 1. [11] 20 min MCAO in rats Striatum 1-7 days [12] 15 min MCAO in hypertensive rats Cortex 0-4 weeks [13] 45 min MCAO in hypertensive rats Cortex 0-4 weeks [18] 30 min MCAO in mice Cortex Unknown [19] 30 min MCAO in rats Striatum 0-5 days [6] Transient 4 vessel occlusion in rats Cortex and hippocampus 1-3 days [15] Photothrombosis in mouse cortex Hippocampus 7-84 days [5] 5 min carotid occlusion in gerbil Hippocampal CA1 2-4 days [3] Ischemic stroke in human cortex Thalamic atrophy by CT 0-12 month [16] Basal ganglia stroke in humans Substantia nigra Unknown [17] MCAO in humans Substantia nigra Unknown [20] Cardiac arrest in humans Hippocampus 1-7 days In the brain area of deepest ischemia, virtually all cells die rapidly by necrosis, resulting in the infarct. In the ischemic penumbra surrounding the infarct, there is a delayed and selective loss of neurons.…”

“…In brain areas distant from the infarct but neuronally connected to the infarct, there is a delayed loss of neurons, known as secondary neurodegeneration. Delayed neuronal death after stroke is potentially more preventable/treatable than the acute neuronal death within the infarct because: (i) the patient has time to reach a hospital and be treated over the time course of this delayed death, (ii) the brain regions with delayed neuronal death are perfused with blood, and therefore drugs can reach the affected cells, (iii) the neurons undergoing delayed neuronal death have probably received less damage/insult than neurons within the infarct, and/or (iv) these neurons may be undergoing forms of cell death that are potentially more amenable to treatment [11,12]. Thus, it is important to understand the mechanisms by which such delayed neuronal death occurs.…”

Curcumin attenuates sensorimotor deficits induced by selective neuronal loss: A study in middle cerebral artery occlusion rat model of transient ischemic stroke.

“…Clinically, TIA is characterized by a transient episode of focal neurological signs which lasts less than 24 hrs and without tissue injury or pannecrosis (acute infarction) 6,7 . TIAs essentially result in a selective neuronal loss (SNL), a patchy (single) neuronal loss with the preserved extracellular matrix and without vascular cell death 6,8 usually injuring the hippocampus's neurons its associated areas, higher cortical regions, and striatum / sensorimotor cortex 9 . The underlying pathophysiology of ischemic stroke (IS) is based on reoxygenation (ischemia-reperfusion) injury 9 .…”