Fibrin is deposited through a disrupted neurovasculature in transgenic mouse models of AD
When the blood–brain barrier is compromised, macromolecules in circulation can accumulate in the brain parenchyma (13
). Because Evans blue dye binds to albumin in the blood, extravasation of the dye serves as a marker for blood–brain barrier permeability and neurovascular damage. We compared Alzheimer's mouse models Tg2576, PDAPP, and TgCRND8 to nontransgenic littermates for defects in the neurovasculature. Because these mice bear AβPP with different familial AD mutations and are driven by different promoters, they exhibit differing ages of onset of Aβ-associated pathology. Therefore, we compared extravasation of Evans blue dye in mice at 6 and 12 mo of age.
As shown in , brains of all three Alzheimer's mouse models were considerably more permeable to the dye. Nontransgenic littermates showed increased blood–brain barrier permeability as age increased. However, in all three Alzheimer's mouse models, the brain was considerably more permeable to the dye at these ages. These data are consistent with previously observed microvascular damage in the Tg2576 mouse (14
), although the TgCRND8 mice show earlier onset.
Figure 1. Blood–brain barrier permeability and neurovascular damage is increased in three mouse models of AD. (A) Evans blue assay indicates increased blood–brain barrier permeability in the Tg2576, PDAPP, and TgCRND8 transgenic mice compared with (more ...)
To visually observe extravascular deposition of Evans blue, mice treated with Evans blue for 6 h were perfused with fluorescent dextran at the time of killing. This 2,000-kD dextran is impermeable to both healthy and damaged blood vessels, and therefore serves as an outline of intravascular space. The nontransgenic littermate blood–brain barrier retained both dyes within the blood vessel as shown in (left). The damaged TgCRND8 blood vessel in (right) showed diffuse accumulation of Evans blue around the contained dextran. As shown underneath each micrograph, the distribution of each fluorochrome can be analyzed for fluorescence intensity across a cross section of a capillary, which reveals Evans blue with a broader distribution than the dextran in the AD mouse (15
). Together with the quantitative extravasation assay, these comprehensive estimates indicated increased blood–brain barrier permeability in the TgCRND8 mouse.
To gain insight into blood vessel health, mice brain sections were stained for platelet/endothelial cell adhesion molecule-1 (PECAM-1). Images of perfused and stained microvasculature were obtained from the cortex of TgCRND8 mice and nontransgenic littermates at 6 mo of age (Fig. S1, available at http://www.jem.org/cgi/content/full/jem.20070304/DC1
). Healthy endothelial cells constitutively express this surface marker (16
), but sections of TgCRND8 brains showed diminished signal intensity, and vessels appeared tortuous and fragmented.
Because the AD mouse blood–brain barrier was permeable to albumin-bound Evans blue dye, we hypothesized that fibrinogen could gain access to the brain's extravascular space. Given this, and because tPA activity is reduced in the AD mouse brain (11
), we reasoned that fibrin could deposit and accumulate over the lifespan of the mouse. Perfused TgCRND8 brains contained elevated levels of fibrin as determined by ELISA. 3–9-mo-old mice showed that Aβ accumulated in an age-dependent manner, and fibrin levels correlated with soluble Aβ1-40
levels, as measured by ELISA from the same tissue homogenates ().
Figure 2. Fibrinogen accumulates through the damaged neurovasculature. (A) Fibrin deposition parallels age-dependent Aβ accumulation. TgCRND8 cortex and hippocampus were isolated, and homogenates were assayed each for fibrinogen and Aβ1-40 by ELISA. (more ...)
Neuroinflammation and microvascular injury are diminished by pharmacologic depletion of fibrinogen
Because fibrin is a proinflammatory molecule and could aggravate pathology upon exiting the vasculature (17
), we asked if fibrinogen depletion might reduce the inflammation in AβPP transgenic mice. TgCRND8 mice are appropriate because of the early onset of neurovascular dysfunction and neuroinflammation, as seen by microgliosis at 13 wk (12
). We therefore reduced circulating fibrinogen levels using a recombinant form of the Malayan pit viper protease ancrod. Ancrod is a thrombin-like protease that cleaves fibrin and prevents its polymerization, allowing degradation by the liver and removal from circulation (19
). TgCRND8 mice were treated with either ancrod or saline for 4 wk before killing at 6 mo of age. Fibrinogen levels were reduced by 50–75% in circulation by ancrod. Accordingly, sections of perfused brains after ancrod treatment showed diminished fibrin immuno reactivity (see ).
Figure 7. Fibrin deposition and vascular damage are modified by manipulation of fibrinogen levels and fibrinolysis. (A) Representative images of perfused brains from each treatment group stained for fibrin. Images were tiled together using a motorized stage on (more ...)
Perfused sections of transgenic brains from each treatment group were stained with CD11b, an integrin receptor present on microglia, and inflammatory foci were visualized. The total area of inflammatory foci can be quantified within the regions of interest, as shown in . Areas of inflammation were compared between ancrod and saline. Ancrod treatment reduced the area of inflammation by ~64% (; P = 0.00001). In saline-treated mice, microglia were identified by their amoeboid morphology. As shown in , these aggregated microglia formed inflammatory foci and appeared to concentrate around plaques with reduced number after fibrinogen depletion. Because ancrod is a protease and could be acting directly on Aβ levels, we quantified levels of plasma Aβ1-40 and cortical Aβ, neither of which were substantially different in ancrod-treated mice (), indicating that depletion of fibrinogen, rather than deposited Aβ, is responsible for the reduced microgliosis.
Figure 3. Fibrinogen depletion and inhibition of fibrinolysis in the 6-mo TgCRND8 mouse have opposite effects on neuroinflammation. (A) Representative immunofluorescent images of brains colabeled for Aβ (left) and CD11b, a marker for activated microglia (more ...)
As inflammation can contribute to blood–brain barrier permeability, we assayed ancrod-treated mice for Evans blue extravasation and found a reduction when compared with saline-treated mice (see ). This attenuation of vascular damage prompted analysis of the microvasculature from both cortex and hippocampus. Identical areas of the brain () were quantified for vascular density as a percentage of image area () and indicated that ancrod treatment partially prevents blood vessel loss.
Figure 4. Fibrinogen depletion and inhibition of fibrinolysis in the TgCRND8 mouse have opposite effects on neurovascular damage. (A) Neurovasculature in ancrod-, saline-, and tranexamic acid–treated TgCRND8 mice. Images of brains labeled for PECAM-1 (black) (more ...)
Neurovascular pathology is promoted by pharmacologic inhibition of fibrinolysis
To complement fibrinogen-depletion experiments, we tested whether absence of plasmin-mediated clearance of fibrin accelerates pathology. We treated TgCRND8 mice with a plasmin inhibitor, tranexamic acid, for 4 wk before killing at 6 mo of age alongside littermates treated with saline or ancrod. Inflammatory foci were again visualized using microglia staining with CD11b (). Plasmin inhibition by tranexamic acid treatment significantly increased microgliosis in treated mice as compared with control mice (P = 0.014; ).
We also reasoned that the inhibition of plasmin-mediated clearance of fibrin and subsequent inflammation could aggravate neurovascular damage in TgCRND8 mice. We observed that administration of tranexamic acid increases damage to the blood–brain barrier (see ). Decreased blood–brain barrier integrity after 4-wk tranexamic acid treatment prompted analysis of the microvasculature. Tranexamic acid–treated TgCRND8 mice showed a reduction in microvascular density, and vessels appeared damaged ().
With the increased pathology observed in the tranexamic acid–treated animals, we asked if inflammation and Aβ were sufficient to promote neurodegeneration. Active caspase-3 staining did not reveal apoptotic cells in treatment or control groups. Samples also were negative for neurodegeneration by Fluoro-Jade B staining (unpublished data).
Neurovascular pathology in the transgenic mouse model is modulated by genetic deficiency in plasminogen or fibrinogen
We crossed transgenic AD mice to mice deficient for fibrinogen (fib−/−
) to obtain TgCRND8;fib+/−
mice bearing only one copy of the fibrinogen gene. Additionally, because accumulated fibrin in the extravascular space can cause damage, we asked if a reduction in plasminogen levels on a background of the AβPP transgene could promote neurovascular pathology. Similar to the fibrinogen cross, we generated TgCRND8;plg+/−
mice and compared them to TgCRND8 littermates. We examined the N1 generation from mice crossed to TgCRND8 mice because pathology presents at an earlier age than PDAPP and Tg2576. Heterozygosity for plasminogen deficiency in TgCRND8 mice produced a significant increase in Evans blue extravasation (; P = 0.042). Conversely, TgCRND8;fib+/−
mice showed reduced neurovascular pathology at 6 mo (P = 0.003). Plg+/−
controls showed little permeability to the dye, suggesting that a product of the AβPP transgene is necessary for neurovascular pathology. To control for the possible effects of different genetic backgrounds on the production and metabolism of the AβPP transgene, PDAPP mice were backcrossed >10 generations onto the C57BL/6 background before crossing with plg−/−
mice, which share the C57 background. The results shown in Fig. S2 (available at http://www.jem.org/cgi/content/full/jem.20070304/DC1
) indicate increased blood–brain barrier pathology in PDAPP;plg+/−
mice when compared with PDAPP littermates, consistent with the results shown in .
Figure 5. Genetic plasminogen and fibrinogen deficiency modulate defects in the AD mouse blood–brain barrier. 6-mo-old AD mice deficient for plasminogen (TgCRND8;plg+/−) and fibrinogen (TgCRND8;fib+/−) were assayed for Evans (more ...)
Because Evans blue dye is fluorescent, the entire hemisphere can be visualized for dye extravasation to determine which areas of the brain are most affected. Images of cerebral hemispheres of each genotype were compared (). The cortex and hippocampus were affected with the highest levels of neurovascular damage, consistent with the observation that hippocampus and cortex show the most Aβ deposition. The data indicated that the removal of one copy of the plasminogen gene accelerates the loss of microvascular integrity in TgCRND8 mice, whereas reduction of one copy of the fibrinogen gene slowed pathogenesis. At 3 mo of age, mice homozygous for plasminogen deficiency showed blood–brain barrier damage (Fig. S3, available at http://www.jem.org/cgi/content/full/jem.20070304/DC1
) but did not show substantial neuroinflammation. Because these mice died early, it cannot be determined if the inflammation would have developed to levels comparable to those of older AD mice. Nonetheless, this finding indicates that the presence of Aβ exaggerates fibrin-related neuroinflammation.
Figure 6. Localization of vascular pathology in AD mice deficient for fibrinogen or fibrinolysis. (A–F) Composite images of cerebral hemispheres of 6-mo-old mice perfused with Evans blue for 6 h. TgCRND8;fib+/− (D) and TgCRND8;plg+/− (more ...)
Fibrinogen depletion protects against the deleterious effects of plasmin inhibition
Suppressing plasmin activity with tranexamic acid leads to increased blood–brain barrier breakdown and inflammation, as shown in and , respectively. As fibrin is the primary target of plasmin proteolysis, this treatment also led to increased fibrin deposition (). However, because plasmin is a potent protease that could have many substrates, we investigated whether the vascular damage and inflammation were due to fibrin accumulation or some other effect of plasmin inhibition. Therefore, we implanted pumps with either saline or ancrod in two groups of mice for a pretreatment period of 1 wk. After pretreatment, both fibrinogen-depleted and control groups received tranexamic acid, to inhibit plasmin activity for 1 wk. All mice were assayed for blood–brain barrier damage and inflammation as before. As expected, plasmin inhibition increased Evans blue extravasation and microglial staining in the control group. By comparison, the fibrinogen-depleted group showed considerably less pathology (). Levels for fibrinogen-depleted mice were similar to those of untreated mice (), suggesting that fibrinogen depletion protected mice from the increased vascular damage and inflammation induced by plasmin inhibition. This result indicates that fibrin deposition is a critical pathologic consequence of reduced plasmin activity in the brains of AD mice.
Figure 8. Ancrod treatment protects AD mice from increased pathology induced by tranexamic acid. (A) Two groups of 6-mo TgCRND8 mice were treated as indicated. (B) Evans blue extravasation. (C) Analysis of CD11b staining. In both cases, pretreatment with ancrod (more ...)