Persistent Angiogenesis in the Autism Brain: An Immunocytochemical Study of Postmortem Cortex, Brainstem and Cerebellum

Abstract

In the current work, we conducted an immunocytochemical search for markers of ongoing neurogenesis (e.g. nestin) in auditory cortex from postmortem sections of autism spectrum disorder (ASD) and age-matched control donors. We found nestin labeling in cells of the vascular system, indicating blood vessels plasticity. Evidence of angiogenesis was seen throughout superior temporal cortex (primary auditory cortex), fusiform cortex (face recognition center), pons/midbrain and cerebellum in postmortem brains from ASD patients but not control brains. We found significant increases in both nestin and CD34, which are markers of angiogenesis localized to pericyte cells and endothelial cells, respectively. This labeling profile is indicative of splitting (intussusceptive), rather than sprouting, angiogenesis indicating the blood vessels are in constant flux rather than continually expanding.

Keywords: CD34; Endothelial; Intussusceptive; Nestin; Pericytes; Superior temporal cortex.

Fig. 1

Pericyte cell labeling in cerebral blood vessels was performed in an 8.5 year. old ASD donor (HSB-4640). All antibodies were mouse monoclonal and were used at 1:1000 dilution. a, b: The label with CD146 showed pericyte cell bodies on the abluminal surface of arteriole vessels. CD146 is a mesenchymal stem cell marker that labels pericytes cells in the brain. No clear labeling of smaller vessels (capillaries) was found. c, d The label with α-SMA showed pericyte cell bodies and fibers on the abluminal surface of arteriole vessels. α-SMA is the contractile protein in smooth muscle and in pericytes. No clear labeling of smaller vessels (capillaries) was found. e, f Vimentin IR of cell bodies and fibers appeared on the abluminal surface of arteriole and capillaries. Vimentin labeled the cell and its processes similar to nestin-positive pericytes. Vimentin is a fibrillary cytoskeletal protein seen in immature pericytes. g, h The label with nestin showed pericyte cell bodies and fibers on the abluminal surface of arteriole vessels. Nestin is a fibrillary cytoskeletal protein expressed in cells that are capable of proliferate. a, c, e, g were photographed with a ×25 (scale bar 50 µm) and × 63 (scale bar 30 µm) objective

Fig. 2

Nestin IR of blood vessels was performed in STC of control 2.1 years. (a, b, c) and ASD donor, age 2.8 years. (d, e). a A low power view showed the nestin-positive arterioles and capillaries in Layer I–VI of the STC in young control. The nestin-positive blood vessels were distributed over all cortical layers. b A view of layer III–V of the STC showed the vessels labeled by the nestin antibody in control (b) and ASD donors (d). Both precapillary arterioles and capillary vessels were labeled. At a higher magnification, the uneven cellular labeling pattern of nestin was apparent in both control (c) and ASD (e) nestin-positive vessels with heavier staining at the junction of the vessels. Nestin-positive glial cells were frequently seen in layer V adjacent to labeled blood vessels in ASD (e) but not NDI controls. Scale bar for a and d = 300 µm and scale bar for c and e is 50 µm

Fig. 3

Age comparison of nestin IR was made between control and ASD donors. Nestin-positive blood vessels were shown in STC from ASD donors ages 2.8 year (B-6399), 8.5 year (HSB-4640), 14.4 year (UMB-4899) and 20.8 year (UMB-4999). In the NKD control donors, nestin-positive blood vessels were seen at age 2.1 year, (BTB-4235), but not at 8.6 year (UMB-1706), 13.7 year, (UMB-1790) or 20.5 year (UMB-4590). All donors were male except for the youngest control brain, which was a female. In the youngest ASD case (2.8 years), nestin-positive labeling was seen in cells in STC layer V. Scale bar 50 µm

Fig. 4

Postmortem sections of the cerebellum (HSB-4640 ASD 8.5 year; UMB-1706 control 8.6 year); midbrain/pons and fusiform cortex (UMB4305 ASD 12.9 year; UNB1790 control 13.7 year) from ASD (a, c, e) and control (b, d, f) donors were reacted with nestin antibody. Nestin-positive blood vessels were seen only in the ASD donors in all regions examined. In the cerebellar cortex, the nestin-positive vessels were seen in molecular (M), Purkinje (P) and granular (G) layers in ASD (a) but not control donors (b). In a midbrain/pons section, nestin positive vessels were seen in the midbrain tegmentum in ASD (c) but not in control (d) donors. In fusiform cortex the nestin-positive vessels were seen in layer IV/V from the fusiform cortex in ASD (e) but not in control donors (f). Scale bar 50 µm

Fig. 5

Endothelial cell labeling was shown in cerebral blood vessels in an 8.5 year old ASD donor (USB-4640). All antibodies were mouse monoclonal and were used at 1:1000 dilutions (a, b). The label with UEA1 showed endothelial cell bodies on the luminal surface of all blood vessels. UEA1, a lectin, is a specific marker of endothelial cells. There was no cross reactivity to pericytes or glial cells (c, d). The label with CD34 showed endothelial cell bodies and processes on the luminal surface of blood vessels. CD34 is a transmembrane glycoprotein involved in cell–cell adhesion and expressed in hematopoietic progenitor cell antigen found in bone marrow and in developing blood vessels in a variety of organs. Scale bar was 50 µm for a, c and 30 µm for b, d

Fig. 6

Age comparison of CD34 IR was compared between control and ASD donors. CD34-positive cells were shown in STC from ASD donors ages 2.8 year (B-6399), 8.5 year (HSB-4640), 14.4 years (UMB-4899) and 20.8 year (UMB-4999) and NKD control donors, ages 2.1 year, (BTB-4235), 8.6 year (UMB-1706), 13.7 year, (UMB-1790) and 20.5 year (UMB-4590). Scale bar 50 µm

Fig. 7

The volume density of antibody labeling was determined using morphometric and stereological methods. The total volumes of IRvessels were measured in the STC (a, b), while the total sum for vessel length using stereology was plotted against age (c). a IR of various vascular antibodies were measured in STC. The graph (a) shows the average total volume of immunocytochemically-labeled profiles in STC for ASD samples relative to control values. The only significant increases were seen for nestin and CD34 antibodies, both of which are indicators of angiogenesis. **p = 0.0157. *p = 0.0385. No other antibody staining showed any significant difference between ASD and control STC. b Nestin IR was measured across brain region (b). The total volume for nestin-IR was divided across brain region between ASD and control. c Vessel length was stereologically measured for coded sections of ASD and control donor STC stained with a specific vimentin antibody. This graph plotted various brains for average vessel length density across age and no significant difference between ASD and control was found