Increased numbers of NFTs in STZ-treated pR5 mice.

<p>Gallyas silver staining (black) of coronal section revealed abundant numbers of flame-shaped NFTs and dystrophic neurites in the amygdala of STZ-treated pR5 mice. In sham-injected pR5 mice, NFTs were rare. No NFTs are found in sham- and STZ-injected wild-type (WT) mice. Quantification of standardized serial sections showed 8-fold increased numbers of NFTs in pR5 mice upon STZ-treatment (**, P<0.001, n = 6). Scale bar, 50 µm.</p

Effects of streptozotocin (STZ) administration in mice.

<p>(<b><i>A</i></b>) STZ injection in both 4 months old wild-type (WT) and pR5 mice induces chronically high blood glucose levels, while levels in sham-injected WT and pR5 control mice are normal (n = 12). Blood glucose levels remained high until mice were analyzed at 6 months of age. (<b><i>B</i></b>) Hematoxylin/eosin staining and (<b><i>C</i></b>) immunofluorescence staining with insulin- (green) and glucagon-specific (red) antibodies of pancreatic islets of Langerhans reveals comparable islet architecture in 6 months old sham-injected WT and pR5 mice. However, insulin producing β-cells are absent from both WT and pR5 islets 60 days after STZ administration, whereas glucagon secreting α-cells remained. Nuclei were counterstained with DAPI (blue). Scale bar, 25 µm.</p

STZ-treatment induces hyperphosphorylation of tau in pR5 and non-transgenic mice, but only in pR5 mice it induced tau insolubility.

<p>(<b><i>A</i></b>) Amygdalae of STZ- or sham-injected wild-type (WT) and pR5 mice were extracted with buffers of increasing stringency and separated by SDS-PAGE for Western blotting. RAB fractions contain soluble species of both transgenic human (triangle) and endogenous murine (asterisk) tau, as shown with HT7 and TAU5, respectively. Phosphorylation site-specific tau antibodies AT8, AT270, AT100, 12E8, PHF-1 and PS422 (for details on antibodies see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007917#pone-0007917-t001" target="_blank">Table 1</a>) reveal increased phosphorylation of transgenic and endogenous tau at multiple sites upon STZ-treatment. In sham-treated controls, tau is slightly more phosphorylated in pR5 than WT mice. GAPDH confirmed equal loading. (<b><i>B</i></b>) RIPA fractions of STZ-treated mice contain higher amounts of both transgenic and endogenous tau than in sham-treated controls. Actin confirmed equal loading. (<b><i>C</i></b>) Insoluble proteins were extracted with formic acid (FA). In STZ-treated mice, HT7 and TAU5 show insoluble transgenic and endogenous tau, whereas no tau is detectable in STZ-treated non-transgenic (WT) or sham-injected non-transgenic and pR5 mice. (<b><i>D</i></b>) Extraction of sarkosyl-insoluble tau from amygdala reveals significant amounts of sarkosyl-insoluble transgenic human tau (HT7) were isolated from STZ-treated, but not sham-injected pR5 mice.</p

Increased tau phosphorylation in STZ-treated pR5 mice.

<p>Immunohistochemistry with phosphorylation site-specific antibodies reveals several AT8-positive neurons in the amygdala of sham-injected pR5 mice at 6 months of age. Whereas in sham-injected pR5 mice most AT8-positive cells lack PS422 staining (open arrowhead), there is a subset of neurons that also stains with PS422 (arrowhead). Similarly, dystrophic neurites were AT8- but not PS422-positive in sham-injected pR5 mice (inset). In contrast, upon STZ injection virtually all tau-containing neurons (arrowhead), and dystrophic neurites (inset) within the amygdala of pR5 mice stained with both AT8 and PS422, as revealed by overlay (yellow). Quantification of AT8 and PS422 double positive cells confirms significantly increased numbers of AT8/PS422 double positive in the amygdala of STZ-treated compared to sham-injected pR5 mice (**, P<0.001; n = 6). Scale bar, 50 µm.</p

Details of tau specific antibodies used in this study.

<p>WB, Western blotting; IHC, immunohistochemistry.</p

Relative alterations in CBF following CCAO, MCAO, during occlusion and following reperfusion for Koizumi vs. Longa at 4 h after reperfusion.

<p>Relative alterations in CBF following CCAO, MCAO, during occlusion and following reperfusion for Koizumi vs. Longa at 4 h after reperfusion.</p

Correlations of relative alterations in CBF following CCAO, MCAO, reperfusion, neurological score, weight loss, age and starting weight to ischemic infarct volume.

<p>Correlations of relative alterations in CBF following CCAO, MCAO, reperfusion, neurological score, weight loss, age and starting weight to ischemic infarct volume.</p

Representative laser-Doppler flowmetry traces of SAH and premature reperfusion

<p>(A) A typical representative laser-Doppler flowmetry during Koizumi’s method of middle cerebral artery occlusion (MCAO), during a 60 min occlusion. Insert: representative resulting ischemic lesion analysed by TTC staining. (B) A typical representative laser-Doppler flowmetry during Longa’s method of MCAO, during a 60 min occlusion. (C) Representative laser-Doppler flowmetry of subarachnoid haemorrhage (SAH) during the Koizumi method of MCAO, with resulting ischemic injury. (D) Representative laser-Doppler flowmetry of false-positive SAH. Inserts: image of the same animal, with a brain bleed resulting from vessel perforation, yet leading to no discernible ischemic injury. (E) Representative laser-Doppler flowmetry showing arbitrary units of CBF falling gradually over the course of a 60 min MCAO occlusion time, conducted via the Koizumi method. (F) Representative laser-Doppler flowmetry indicating premature reperfusion, due filament movement out of place, followed by immediate recovery of MCAO by repositioning the filament. Perfusion Units (PU) are arbitrary units of cerebral blood flow.</p

Relative alterations in CBF following CCAO, MCAO, during occlusion and following reperfusion, for thin and thick filaments at 30 min, 4 h, 12 h, and 24 h after reperfusion.

<p>Relative alterations in CBF following CCAO, MCAO, during occlusion and following reperfusion, for thin and thick filaments at 30 min, 4 h, 12 h, and 24 h after reperfusion.</p

Relative alterations in CBF following CCAO, MCAO, during occlusion and following reperfusion for 15 min, 30 min, 45 min and 60 min occlusions measured at 4 h or 24 h after reperfusion.

<p>Relative alterations in CBF following CCAO, MCAO, during occlusion and following reperfusion for 15 min, 30 min, 45 min and 60 min occlusions measured at 4 h or 24 h after reperfusion.</p