Antibodies to the α1-adrenergic receptor cause vascular impairments in rat brain as demonstrated by magnetic resonance angiography

Abstract

Background: Circulating agonistic autoantibodies acting at G protein-coupled receptors have been associated with numerous sever pathologies in humans. Antibodies directed predominantly against the α(1)-adrenergig receptor were detected in patients suffering from widespread diseases such as hypertension and type 2 diabetes. Their deleterious action has been demonstrated for peripheral organs. We postulate that antibodies to the α(1)-adrenergig receptor are relevant pathomolecules in diseases of the central nervous system associated with vascular impairments.

Methodology/principal findings: Using a rat model we studied the long-term action of antibodies against the α(1)-adrenergig receptor either induced by immunization with a receptor peptide or applied by intravenous injection. The vasculature in the rat brains was investigated by time-of-flight magnetic resonance angiography using a 9.4 Tesla small animal MR imaging system. Visual examination of maximum-intensity-projections (MIPs) of brain angiographs revealed the development of vascular defects in antibody- exposed animals between three and eight months of treatment. Relative vascular areas were derived from representative MIP image sections by grayscale analysis and used to form an index of vascular circulation. Animals exposed to the action of α(1)-adrenergig receptor antibodies showed significantly reduced vascular areas (p<0.05). Calculated index values indicated attenuated blood flow in both antibody-treated cohorts compared to their respective controls reaching with (relative units ± standard error, n = 10) 0.839 ± 0.026 versus 0.919 ± 0.026 statistical significance (p<0.05) for peptide-immunized rats.

Conclusion/significance: We present evidence that antibodies to the α(1)-adrenergig receptor cause cerebrovascular impairments in the rat. Our findings suggest the pathological significance of these antibodies in pathologies of the human central nervous system linked to impairments of brain vasculature such as stroke and dementia.

Figure 1. Analysis of rat sera for the presence of antibodies to the α₁-AR.

Antibodies were detected by ELISA. (PEP) rats immunized with the α₁-AR peptide, (C-PEP) controls treated with vehicle. Blood samples were taken about one month before the final TOF-MRA. The obtained sera were analyzed at a 1∶50 dilution.

Figure 2. Anatomical images and TOF-MRA of the rat brain.

Maximum-intensity-projections (MIP) of the TOF-MRA image data are shown for a coronal view (B) and axial view (E) together with corresponding 2-dimensional anatomical T₂-weighted image slices (A, D). C) sagittal MIP view. F) double-oblique MIP view from the left posterior perspective (shown at a smaller scale).

Figure 3. Two-dimensional maximum-intensity-projections of

TOF-MRA of rat brain. Coronal images of rat brains were screened in 3 mm slices. The rat shown in A, B was immunized with the α₁-AR peptide, the rat shown in C, D obtained vehicle as control. Injections were performed monthly. Angiographs of the same animal were taken at the beginning of the treatment (A, C) and eight months later (B, D). Light contrasts correlate with vascular blood flow. Eight months of treatment with the α₁-AR peptide led to apparent attenuations of blood flow (B, right part of brain section) compared to the initial situation (A). Control injections did not result in comparable defects in the same time frame (D versus C).

Figure 4. TOF-MRA of rat brain.

Three-dimensional coronal views of the frontal brain segment. The rat shown is as in Figure 3 which was immunized with the α₁-AR peptide. Angiographs of the same animal were taken at the beginning of the treatment (A, C) and eight months later (B, C). C, D show the same brain segment as in A, C but vertically rotated by 45°. The defects in blood flow in the right part of the brain as displayed in Figure 3 are obvious (B, D) compared to the situation eight months before (A, C).

Figure 5. Black and white threshold images.

Two-dimensional sections of maximum-intensity-projections (MIP) of TOF-MRA images were processed as described in Materials and Methods. The black and white images were generated using the median plus two standard deviations as threshold value. White areas correspond approximately to the area of vessels with blood flow. Exemplarily shown is one animal treated with intravenous injections of the α₁-AR antibody (A, B) and one control animal (C, D) at measurement 1 (A, C) and at measurement 2 eight months later (B, D). The reduction of the white areas in B compared to A is evident.

Figure 6. Box plot of relative brain vascular blood flow.

Vascular area was calculated from two-dimensional section of angiographs of rat brains by grayscale analysis as described in the methods section. Relative vascular circulation of the individual animal is expressed as ratios of vascular areas from the last and the first TOF-MRA. Time between measurements was eight months. Ratios <1 indicate attenuated vascular blood flow. Data are given of animals treated with intravenous injections of the antibody to the α₁-AR (AB; n = 9), their controls (C-AB); n = 10), and rats injected subcutaneously with the α₁-AR peptide (PEP; n = 10) and the respective controls (C-PEP; n = 10). Asterisk indicates statistically significant differences with p<0.05.