Rapid reversible changes in dendritic spine structure in vivo gated by the degree of ischemia

Abstract

Current therapeutic windows for effective application of thrombolytic agents are within 3-6 h of stroke. Although treatment can improve outcome, it is unclear what happens to synaptic fine structure during this critical period in vivo. The relationship between microcirculation and dendritic spine structure was determined in mouse somatosensory neurons during stroke. Spines were, on average, 13 mum from a capillary and were supplied by approximately 100 red blood cells per second. Moderate ischemia (approximately 50% supply) did not significantly affect spines within 5 h; however, severe ischemia (<10% supply) caused a rapid loss of spine and dendrite structure within as little as 10 min. Surprisingly, if reperfusion occurred within 20-60 min, dendrite and spine structure was mostly restored. These data suggest that the basic dendritic wiring diagram remains mostly intact during moderate ischemia and that affected synapses could potentially contribute to functional recovery. With severe ischemia, markedly deformed dendritic structure can partially recover if reperfusion occurs early.

Figure 1.

ET-1 effect on neuronal viability and blood flow. A, Fluorojade staining showing that neurons were dying 24 h after ET-1 injection in a wild-type mouse (urethane anesthesia). Note that, to use fluorojade staining (green) to monitor dying cells, a wild-type C57BL/6 was used. B, Regional blood flow recorded by laser Doppler indicates little effect of preinjection drilling and a significant reduction 15 min after ET-1 injection (∼45 min since the start of injection). C, Dorsal view of the micro vasculature (maximal intensity projection) from 100 planar scans acquired every 2 μm before and after ET-1 injection showing a modest effect of ET-1 on vessel structure. d, Z-projection showing a region from which capillary flow (red line) was recorded, from which spine structure (yellow box) was assessed in f. e, Line-scan data for blood flow in the capillary shown in D before and 1 and 5 h after ET-1 injection. ET-1-induced ischemia leads to reversals in flow direction in the right arm of the large vessel shown in D and g; a T-junction is present. The RBC velocity was 2355 ± 164, 1586 ± 78, and 1267 ± 67 μm/s for pretreatment, 1 h and 5 h, respectively, for the left arm of the large vessel, and the flux was 153, 128, and 91 RBCs per second for pretreatment, 1 h and 5 h, respectively, for the left arm. The RBC velocity was 2355 ± 164, 735 ± 40, and 506 ± 55 μm/s for pretreatment, 1 h and 5 h, respectively, for the right arm of the large vessel, and the flux is 153, 63, and 32 RBCs per second for pretreatment, 1 h and 5 h, respectively, for the right arm. F, Spines (arrowheads) remained stable within 20 μm of where blood flow was reduced (yellow box in D). g, Illustration of RBC flow in the capillary shown in E. Pre, Pretreatment.

Figure 2.

Acute effect of moderate ET-1-induced ischemia on dendritic structure. a, b, Injection of PBS (control) (A) or endothelin (B) does not alter dendritic structure monitored during 5 h of acute imaging. C, Relative change in spine number (compared with pretreatment) over 5 h of ET-1 or PBS treatment (p > 0.05 ANOVA; n = 5 animals for PBS; n = 6 animals for ET-1). D, Image overlay indicates no major changes in spine structure with 5 h of ET-1 treatment. Pre, Pretreatment.

Figure 3.

Effect of RB-induced severe ischemia on brain structure. A, Images from a YFP mouse showing the vasculature and YFP (maximal intensity projection of the first 100 μm of the cortex) before and 30 min after photoactivation (1 min) of RB. Vessels are labeled with Texas Red dextran. b, c, Quantification of spine number and blood-flow velocity for the animal shown in a. d, Dendritic structure (green) was completely lost, and no spines were apparent in this example of RB-induced ischemia with zero blood flow and no reperfusion. Apparent clotting and breakdown of capillaries (red) were seen 30 min after photoactivation of RB. e, In vivo time-lapse images from another animal showing partial recovery of dendrites and spines with confirmed reperfusion. Arrowheads show an example of a spine initially lost that recovered with reperfusion. f, A dendritic region from the yellow box region in E is rotated and overlaid to show the position of the recovered spine. The arrowhead shows the spine indicated in E that recovered at the same place. The arrow shows a spine not recovered after reperfusion. Quantification of spine number (g), blood-flow velocity (h), and flux for this animal (i). Pre, Pretreatment.

Figure 4.

Spine loss and recovery with severe transient ischemia and reperfusion. A, Line scan through a section of capillary shown in C and D to monitor blood flow. Blood flow stops 10 min after photoactivation of RB. B, Spine number expressed as percentage of pre-RB photoactivation (average from 5 regions). c, In vivo images of a dendritic region showing beading and recovery with reperfusion (perfusion can be assessed by the striped image of the capillary that reflects moving RBCs). D, Higher-magnification view of the boxed region in C showing spine loss with ischemia and recovery with reperfusion. Both the dendrites (YFP) and the vessels (FITC-dextran) were labeled with the same color. Arrowheads show examples of spines initially lost and recovered with reperfusion. Arrows indicate RB-induced clotting in a section of a capillary that resolved spontaneously over a 2 h period after photoactivation. Pre, Pretreatment.