Peripheral injections of Freund's adjuvant in mice provoke leakage of serum proteins through the blood–brain barrier without inducing reactive gliosis

Abstract

Breakdown of the blood–brain barrier (BBB) and ensuing gliosis are common events following physical trauma to the central nervous system (CNS) or during autoimmune diseases such as experimental allergic encephalomyelitis (EAE). Some studies of EAE in rodents report that peripheral injections of complete Freund's adjuvant (CFA), which contains heat-inactivated Mycobacterium to provoke peripheral inflammation without adversely affecting the CNS, can itself lead to increased BBB permeability to small tracer molecules and certain serum proteins. To study the equivocal relationship between serum protein extravasation and reactive gliosis, we injected C57BL/6 mice with CFA and histologically assessed the permeability of various serum proteins and the reactivity of proximal microglia and astrocytes in the uninjured brainstem and spinal cord enlargements after 1–4 weeks. Our results confirm that CFA injections induce progressive increases in the perivascular extravasation of serum IgG, albumin, IgM, and exogenous horseradish peroxidase, all to varying degrees, most prominently in the brainstem and cervical spinal cord after 2–3 weeks. More importantly, neither microglial cells nor astrocytes in regions of focal serum protein leakage appeared morphologically reactive based on immunoreactivity for CR3 receptors (Mac-1) or glial fibrillary acidic protein (GFAP), respectively. Because we found no evidence of T cell infiltration accompanying the exudates, our results indicate that in the absence of physical trauma or inflammatory cells resident CNS neuroglia remain quiescent upon exposure to extravasated serum proteins.

Introduction

The central nervous system (CNS) has long been considered an `immunologically-privileged' site based, in part, on the existence of a functional blood–brain barrier (BBB) constituted by endothelial cells which limit the passage of solutes and cells from the blood. In this regard, the critical steps in the initiation of inflammatory processes in the CNS parenchyma are perturbations in the structural integrity of the BBB and the recruitment of blood-borne cells, most notably T cells [23]. A common feature of CNS inflammation induced by either physical trauma, neurodegeneration, or direct injection of proinflammatory agents is the systematic association of serum protein extravasation through the BBB in the affected regions with the activation of resident neuroglial cells leading to reactive gliosis 1, 2, 3, 4, 14, 26, 40, 50, 55. Although these events appear to occur concomitantly, the cellular consequences of exposure to extravasated serum proteins in vivo remains unclear due to the myriad of secondary effects stemming from the pathology of CNS insults.

Experimental allergic encephalomyelitis (EAE) is a cell-mediated autoimmune disease of the rodent CNS that is associated with breakdown of the BBB, inflammatory cell recruitment and reactive gliosis 15, 17, 27, 28, 33. The disease is generally induced by peripheral injections of CNS tissue homogenized with complete Freund's adjuvant (CFA) which contains heat-inactivated Mycobacterium tuberculosis[43]. It is common practice to use CFA injections alone as a control for EAE because this leads to peripheral inflammation without the development of clinical signs or cellular inflammatory responses in the CNS 12, 35, 53. Nevertheless, studies have shown that rodents immunized with CFA alone display increased BBB permeability to blood-borne tracers and certain serum proteins 39, 60, indicating that peripheral inflammation can provoke leakage of the BBB. Therefore, to study the relationship between serum protein extravasation and reactive gliosis in the absence of CNS inflammation, we peripherally injected mice with CFA and examined whether exposure to serum proteins per se is sufficient to induce the morphological activation of microglial cells and astrocytes.