Gold nanoparticle-enhanced X-ray microtomography of the rodent reveals region-specific cerebrospinal fluid circulation in the brain

Abstract

Cerebrospinal fluid (CSF) is essential for the development and function of the central nervous system (CNS). However, the brain and its interstitium have largely been thought of as a single entity through which CSF circulates, and it is not known whether specific cell populations within the CNS preferentially interact with the CSF. Here, we develop a technique for CSF tracking, gold nanoparticle-enhanced X-ray microtomography, to achieve micrometer-scale resolution visualization of CSF circulation patterns during development. Using this method and subsequent histological analysis in rodents, we identify previously uncharacterized CSF pathways from the subarachnoid space (particularly the basal cisterns) that mediate CSF-parenchymal interactions involving 24 functional-anatomic cell groupings in the brain and spinal cord. CSF distribution to these areas is largely restricted to early development and is altered in posthemorrhagic hydrocephalus. Our study also presents particle size-dependent CSF circulation patterns through the CNS including interaction between neurons and small CSF tracers, but not large CSF tracers. These findings have implications for understanding the biological basis of normal brain development and the pathogenesis of a broad range of disease states, including hydrocephalus.

Introduction

Postnatal brain development involves dramatic changes in the cells and structure of the brain and the establishment of interconnected signaling pathways with newborn neurons arising from the subventricular zone (SVZ) and hippocampus¹. These neural precursor cell populations are adjacent to the cerebrospinal fluid (CSF) spaces and are thought to be modulated by CSF constituents that change during development^2,3,4,5,6. CSF has been shown to promote the function and survival of glia and neurons in vitro and may have a role in modulating neuronal activity^{5,7,8,9,10,11,12}.

CSF circulation has recently received renewed interest, in part due to the newly described pathways for fluid movement via the glymphatic and lymphatic pathways and their putative role in neurodegenerative diseases such as Alzheimer’s Disease and multiple sclerosis^13,14,15. These routes for CSF movement have primarily been studied in adult and senescent animals in relation to waste removal. The understanding of CSF as a source of nutrient and growth factor delivery in the developing brain is still evolving.

In fact, our current understanding of CSF circulation is not representative of the conditions in developing animals, calling into question its relevance to some of the most common CSF disorders of childhood, such as hydrocephalus. For example, the historically implicated structures for CSF handling along the superior sagittal sinus, arachnoid villi, and granulations, are not fully developed in humans until two years of age and are not present in a number of species, including rodents^16,17,18. In addition, while sparse meningeal lymphatics have been observed in mice at birth around the foramen magnum and pterygopalatine artery¹⁹, most functional meningeal lymphatic connections take three to four weeks to fully develop postnatally^19,20. Finally, glymphatic CSF handling has primarily been studied in adult animal models (with a few exceptions^21,22,23,24), and most studies have focused on the superficial cortical surface adjacent to the middle cerebral artery (MCA) as it can be visualized with two-photon live imaging¹³. CSF clearance via the Na⁺/K⁺/Cl⁻ cotransporter 1 (NKCC1) during postnatal development was recently reported²⁵, however, this and other components of putative CSF movement, including lymphatic CSF outflow, glymphatic fluid and solute handling, perineural, perivenous, and spinal subarachnoid space (SAS) CSF handling, have been studied largely in isolation from one another^15,26,27,28. Few, if any, prior analyses of CSF dynamics have simultaneously been able to examine the cerebrum, cerebellum, and spinal cord due to imaging constraints, a limitation that hampers our ability to understand global CSF circulation throughout the central nervous system (CNS).

Special considerations for the study of CSF in the developing brain include the concern that disruption of CSF dynamics interrupts normal brain development^29,30,31,32. More fundamental is the enigma surrounding the directionality of the relationship between CSF accumulation and neurodevelopmental aberrations²⁹. Specifically, CSF circulation has largely been thought of in the context of secretion and drainage without consideration for how CSF (and its solutes) interfaces with specific populations of developing neurons and glia in the CNS. In this present study, we address how CSF interacts with the brain and spinal cord by utilizing a CSF imaging method, gold nanoparticle-enhanced X-ray microtomography (AuNP-XRM), for high-resolution 2-D and 3-D imaging of CSF pathways through the entire CNS of neonatal rats with histologic confirmation^33,34,35. We present a comprehensive descriptive map of differential CSF influx to intraparenchymal functional-anatomic targets. Small CSF tracers travel via previously uncharacterized influx pathways from the basal cisterns to deep functional-anatomic cell groupings, and from the SAS to the cerebral and cerebellar cortices. Large CSF tracers enter the extracellular space within the hippocampus and septal area via transventricular, transependymal, and transleptomeningeal routes, contrasting the small CSF tracer targets, which are primarily neurons and functional-anatomic cell populations. CSF delivery to these regions is altered in a model of known CSF pathology^36,37, intraventricular hemorrhage-post-hemorrhagic hydrocephalus (IVH-PHH), with implications for mechanisms of brain injury and impaired brain development in PHH.

Results

CSF circulation imaging using AuNP-XRM

To image all pathways for CSF movement within the CNS, we utilized high resolution XRM with AuNPs as a CSF tracer (Fig 1a)^33,34,35. Polyethylene glycol (PEG)-coated AuNPs constituted in artificial CSF (aCSF) were injected into the CSF spaces as a contrast agent and CSF tracer. Following injection, samples were prepared for XRM, as shown in Figs. 1a, b (also see “Methods”). The use of this method resulted in high resolution (14.7 μm/pixel) images of the entire brain and spinal cord in which the CSF spaces and parenchyma including discrete nuclei (cell groupings), tracts, and cranial nerves could be clearly visualized. The AuNPs have high X-ray attenuation, low negative zeta potentials (−1.3 ± 0.4 mV for 1.9 nm AuNPs and −1.6 ± 0.8 mV for 15 nm AuNPs), and average hydrodynamic diameters of 3.79 ± 0.16 and 35.91 ± 0.62 nm for 1.9 and 15 nm AuNPs, respectively….