Elsevier

NeuroToxicology

Volume 33, Issue 3, June 2012, Pages 436-443
NeuroToxicology

FLA
In vivo administration of fluorescent dextrans for the specific and sensitive localization of brain vascular pericytes and their characterization in normal and neurotoxin exposed brains

https://doi.org/10.1016/j.neuro.2012.04.004Get rights and content

Abstract

We have aimed to develop novel histochemical markers for the labeling of brain pericytes and characterize their morphology in the normal and the excitotoxin-exposed brain, as this class of cells has received little attention until recently. Pericyte labeling was accomplished by the intracerebroventricular injection of certain fluorescent dextran conjugates, such as Fluoro-Gold-dextran, FR-dextran, FITC-dextran and Fluoro-Turquoise (FT)-dextran. 1–7 days after the tracer injection, extensive labeling of vascular pericytes was seen throughout the entire brain. These cells were found distal to the endothelial cells and exhibited large dye containing vacuoles. The morphology of the pericytes was somewhat variable, exhibiting round or amoeboid shapes within larger intracellular vesicles, while those wrapping around capillaries exhibited a more elongated appearance with finger-like projections. The use of FG-dextran resulted in bluish yellow fluorescently labeled pericytes, while FR-dextran resulted in red fluorescent labeled pericytes, FITC-dextran exhibited green fluorescent pericytes and FT-dextran showed fluorescent blue pericytes in the brain. We have used these tracers to study possible changes in morphology and pericyte number following kainic acid insult, observing that the number of pericytes in the injured or lesioned areas of the brain is dramatically reduced compared to the non-injured areas. These novel fluorochromes should be of use for studies involving the detection and localization of pericytes in both normal and pathological brain tissues.

Highlights

► Fluorescent dextrans when injected into the lateral ventricle of the brain labeled perivascular pericytes. ► Using these fluorescent dextrans, one can determine the impact of various neurotoxicants on pericyte distribution in the brain. ► Also following neurotoxicant exposure, the pericyte morphology was different compared to saline treated control animals.

Introduction

Pericytes were named Rouget cells after French scientist Charles Marie Benjamin Rouget (Rouget, 1874). Broadly, pericytes are polymorphic, elongated, contractile and multi-branched peri-endothelial cells that vary in their degree of development in the microvasculature. These cells are located abluminal to the endothelial cells and luminal to the parenchymal astrocytes. Morphologically, they have round nuclei and long processes which encircle the endothelial wall (Mandarino et al., 1993). Pericytes serve to support blood vessels but they can differentiate into fibroblasts, smooth muscle cells or macrophages when required (Balabanov and Dore-Duffy, 1998). Recently, it has been proposed (von Tell et al., 2006) that pericytes play important roles in blood–brain barrier stability and angiogenesis as these pericytes express smooth muscle actin and desmin, two crucial proteins present in the smooth muscle cells. Also, their contact with the endovascular cells makes them ideal candidates for blood flow regulation at the capillary level (Dore-Duffy, 2008). Pericytes may communicate with neurons, astrocytes and endothelial cells of the brain by direct physical contact and through the autocrine and paracrine pathways (Balabanov and Dore-Duffy, 1998). A body of evidence suggests that pericytes serve macrophage activity in the brain (Dore-Duffy, 2008), and also upregulate the macrophage activity in response to traumatic brain injury (Dore-Duffy et al., 2000), bacterial infection and other diseases (Gerhardt and Betsholtz, 2003). Therefore, pericytes are well equipped to respond to changes in the micro-environment.

The following three types of pericytes can be characterized based on their microvascular location: (a) pre-capillary arterioles, (b) capillaries, and (c) post-capillary venules (Shepro and Morel, 1993). Developmentally, specific pericyte precursor cells have not been clearly identified and their origin is controversial. It has been proposed that one type of pericytes is of mesodermal origin (Vorbrodt and Wisniewski, 1982). Another group of authors suggest that pericytes are derived from multipotent aortic angioblasts (Minasi et al., 2002), whereas others suggest that pericytes are derived from neural crest cells depending on their site in the embryo (Bergwerff et al., 1998, Carmeliet, 2004, Korn et al., 2002). Korn et al. suggested a neuro-ectodermal origin of brain pericytes and used the expression of muscle actin as a pericyte marker (Korn et al., 2002).

Positive identification of pericytes in the brains is hindered by the lack of a specific marker. A body of evidence suggests that pericytes express surface antigens, such as CD13, α-smooth muscle actin (SMA), desmin, vimentin and NG-2. However, the specificity of these antibodies to stain pericytes is dubious as these markers not only stain pericytes but also have an affinity for other structures which contain above mentioned surface antigens. Pericytes also express markers of the monocyte/macrophage lineage such as CD 11b/ED1 (Balabanov and Dore-Duffy, 1998, Graeber et al., 1989), but CD11b also stains activated microglia following toxic insults to the organism. The pericytes in the lung also express inducible nitric oxide synthase (iNOS) during cytokine insult, suggesting a role in hemodynamics (Kim et al., 2008).

In the present study, we have tested four different fluorochrome-labeled dextrans for their ability to localize pericytes in the brain. Previous studies have demonstrated that Fluoro-Gold can be used as a fluorescent retrograde axonal tracer (Schmued and Fallon, 1986), which will label the vesicles, plasma membrane and the nucleolus of retrogradely labeled neurons. In this connection, it is noteworthy that Fluoro-Gold was shown to be taken up by perivascular cells after both intracerebral and intraneural injections (Pennell and Streit, 1998, Streit and Graeber, 1993). However, Fluoro-Gold conjugated with dextran (FG-dextran) has the ability to stain vascular pericytes in the brain when injected into the lateral ventricle. A second fluorochrome, TRITC dextran (Fluoro-Ruby; FR) that serves both anterograde as well as retrograde axonal transport, also has the ability to stain the brain vascular pericytes when injected into the lateral ventricle of the brain. We have developed two additional fluorochromes; specifically FT-dextran and FITC-dextran which result in blue and green fluorescent labeled pericytes, respectively, following injection into the lateral ventricle. We have compared the morphology of these pericytes in the normal animal versus animals exposed to kainic acid, an NMDA agonist. We hypothesize that this neurotoxicant may alter the structure and function of pericytes in brain regions vulnerable to neurotoxicants.

Section snippets

Animals

Experiments were performed on adult male Sprague–Dawley rats (NCTR breeding colony) weighing 400–450 g. The animals were housed under standard environmental conditions (light between 06.00 and 18.00 h, temperature 22 ± 1 °C, with ad libitum access to water and rat chow). All experimental protocols were approved by the NCTR IACUC.

Animal preparation and fluorescent dextran infusion

Five days prior to sacrifice, animals were anesthetized with ketamine and xylazine (80/11 mg/kg body weight) and one of the aforementioned labeled dextran dyes was pressure

Pericyte distribution and morphology in the normal rat brain

Administration of 10% FR into the lateral ventricle of the rat brain showed red labeled perivascular pericytes predominately surrounding the capillaries (Fig. 1A). The lumen of the capillaries is bordered by endothelial cells as seen after FT-Gel perfusion and the pericytes surround their abluminal surface (Fig. 2G). In the normal animals, normal pericytes have been found around the capillaries (Fig. 1A). These pericytes have prominent nuclei and are broad flat cells with slender projections.

Discussion

Since the original discovery of pericytes by Rouget (Rouget, 1874), their nature has generated much controversy as reflected by the conflicting publications. Pericytes have been described as (i) a contractile cell which encircles the capillary, (ii) a branching contractile cell on the abluminal wall of a capillary, (iii) an elongated contractile cell enmeshed within the pre-capillary arterioles, (iv) a relatively undifferentiated connective tissue cell in the capillaries or other small blood

Conclusions

  • a)

    In conclusion, these four novel fluorochrome dextran conjugates were shown to be useful for the detection, and localization of pericytes in the brain under normal as well as in pathological conditions.

  • b)

    The loss of proper contact between pericytes and astrocyte or endothelial cells can result in pericyte dysfunction and development of diseases, such as stroke (Duz et al., 2007), multiple sclerosis (Bolton, 1997, Claudio and Brosnan, 1992), early diabetic neuropathy (Pfister et al., 2008, Shojaee

Conflict of interest

There is no conflict of interest.

Acknowledgment

This work was supported by FDA Protocol E 7312.
DISCLAIMER

The contents of this manuscript do not necessarily reflect the views and policies of the U.S. Food and Drug Administration, nor does the mention of trade names or commercial products constitute endorsement or recommendation for use.

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