Hydrocephalus is a common complication after aneurysm subarachnoid hemorrhage (SAH) [1]. According to the classical bulk flow theory, cerebrospinal fluid (CSF) flows through the fourth ventricle outlets into the subarachnoid space around the brain and the spinal cord before it is finally reabsorbed the venous system through arachnoid granulations (AGs) in the sagittal sinus. The blockage of AGs by red blood cells and their decomposition products causes CSF absorption disorder and ultimately results in chronic hydrocephalus [2]. However, there is still no definite quantitative experiment to confirm CSF absorption by AGs. In addition, some researchers believe that CSF flows into the extracranial lymphatic system through lymphatic vessels associated with the extracranial segments of cranial nerves [3]. Research on the circulation pathways of CSF has mainly been conducted on rats and dogs, which is unable to fully explain human CSF circulation. As AGs differ hugely in different species and different developmental stages, we believe that the reabsorption pathways of CSF are diverse and of different significances. We speculate that animals with a developed sense of smell mainly rely on the powerful extracranial lymphatic system to absorb CSF, while primates mainly rely on AGs. Previous researchers have used various experimental animals to study the CSF circulation pathways, but differences in species have led to different experimental results [4]. Based on our experimental conditions, preliminary research, and pertinent literature, we selected cynomolgus monkeys as experimental subjects, which are more similar to humans in anatomy, hoping to draw more accurate conclusions. In this research, we established hydrocephalus primate models after SAH by repeatedly injecting autologous blood into the subarachnoid space of cynomolgus monkeys to observe the ultrastructural changes of AGs under pathological conditions, in hopes of providing a theoretical basis for the etiology and prevention of hydrocephalus after SAH.

First of all, head CT examination results performed on normal cynomolgus monkeys showed that their bilateral cerebral hemispheres’ size, shape, and location were normal, with no apparent abnormal density shadows in the brain parenchyma. The shape, size, and location of their ventricles were normal. Their brain cistern and sulci were not enlarged (Fig. 1A). Simultaneous brain MRI scans showed no abnormal signals (Fig. 1B,C). HE staining of normal cynomolgus monkey intracranial tissues showed that the normal arachnoid granular structure exists in the superior sagittal sinus. Its structure and position are similar to those of humans (Fig. 2A,B). The endothelial cells in the outermost layer of normal AGs and layered fibrocytes and connective tissue fibers, were observed under an electron microscope (Fig. 2C,D).

Fig. 1.

Representative imaging of normal cynomolgus monkeys. (A) Head CT shows normal. No mass, hemorrhage, or hydrocephalus. Basal ganglia and posterior fossa structures are normal. No major established vessel vascular territory infarction. No intra- or extra-axial collection. The basal cisterns and foramen magnum are patents. (B,C) Brain MRI shows normal. No abnormal focal areas of altered signal intensity in the cerebral hemispheres, brainstem, or cerebellum. The appearance and intensity of brain parenchyma are normal. The ventricular system and cisternal spaces appear normal. No evidence of intracranial space occupying lesion or apparent vascular anomaly is detected. There is no shift of the midline structures.

Fig. 2.

Ultrastructure of normal AGs. (A,B) HE staining shows that AGs composed of endothelial cells protrude into the sinus lumen. The endothelial cells in the outermost layer of normal AGs and layered fibrocytes and connective tissue fibers, are present under a transmission electron microscope. (C) Endothelial cells and adjacent fibroblasts in the outermost layer of AGs from the transverse sinus of normal cynomolgus monkeys. (D) Endothelial cells and adjacent fibroblasts of AGs from the superior sagittal sinus of normal cynomolgus monkeys. DM, dura mater; AG, Arachnoid Granulation; CF, collagen fiber; EC, endothelial cell; Double arrow, the thickness of CF; (A,B) 40$\times$; (C,D) Scale bar = 2 $\mu$m.

After confirming the location and structure of cynomolgus AGs, we began to prepare primate models of SAH. After receiving repeated autologous blood injections into the subarachnoid space through the cisterna magna, cynomolgus monkeys gradually showed signs of movement disorder (limb weakness and movement decrease), anorexia, and unresponsiveness. The weights of the monkeys were lowered to 2.78 to 3.63 kilograms. Then head CT scan showed high-density shadows in the subarachnoid space (Fig. 3C), and both head CT and MR (Fig. 3D) showed dilation of the ventricular system. The imaging results above suggested the formation of hydrocephalus after SAH. Hydrocephalus monkeys were dissected, and the subarachnoid space was congested with blood clots (Fig. 4). The arachnoid granular tissues of the monkeys with hydrocephalus were observed under a transmission electron microscope. The outer arachnoid was filled with red blood cells (Fig. 5A). Condensation and marginalization of some nucleus chromation were observed, part of the mitochondria and endoplasmic reticulum were swollen with vacuoles, apoptosis and necrosis were found in the endothelial cells of AGs, and the connective tissue scaffold around the cells was disordered and thickened. Collagen fiber hyperplasia was observed, and the fibrous connective tissue of the outer arachnoid layer was thicker than normal (Fig. 5B–D). For a more intuitive comparison, we measured the CF thicknesses of normal and pathological AGs. We found that CF in normal AGs (Fig. 5E,F) is scattered, and the thickest part measures 2 $\pm$ 0.54 $\mu$m, while in the AGs of monkeys suffering hydrocephalus after SAH, CF thickness is about 4 $\pm$ 1.23 $\mu$m. All these signs suggested that AGs suffered extensive fibrosis.

Fig. 3.

Imaging of cynomolgus monkeys before and after blood injection. (A,B) Both head CT and Brain MRI show normal. (C) After injecting autologous blood through the occipital cistern, the head CT of cynomolgus monkeys shows high-density shadows in the subarachnoid space (black arrows). (D) After the injection, MRI of cynomolgus monkeys shows dilation of the ventricular system.

Fig. 4.

Monkeys were dissected, and the subarachnoid space was congested with blood clots. (A) Blood clots under the dura. (B) Blood clots full of sulci and gyri.

Fig. 5.

Ultrastructure of AGs after SAH in cynomolgus monkeys. (A) The outer arachnoid is filled with red blood cells (indicated by a black arrow). (B–D) The fibrous connective tissue of the outer arachnoid layer appears thicker than normal, and AGs show extensive fibrosis. (E,F) CF in normal AGs is scattered. (A) Scale bar = 5 $\mu$m, (B–D) Scale bar = 2 $\mu$m, (E,F) Scale bar = 1 $\mu$m.

AGs, also known as arachnoid villi and pacchionian granulations, are small protrusions of the arachnoid mater into the outer membrane of the dura mater. They protrude into the dural venous sinuses of the brain and allow CSF to pass from the subarachnoid space into the bloodstream. AGs were first discovered by the Italian scholar Antoine Pacchion in the 18th century [5]. Later studies confirmed that AGs have begun to form in the superior sagittal sinus, parasagittal sinus, and transverse sinus within one to two months before birth, which gradually develops under the permanent pressure of CSF after birth. Human AGs are mainly distributed in the superior sagittal sinus (53%), especially where the central vein joins the superior sagittal sinus; followed by the transverse sinus (28%), especially where the vein of Labbe merges into the transverse sinus; some in the straight sinuses (17%); and the rest in the intracranial venous sinuses [6, 7]. Conegero et al. [8] used scanning electron microscopy to find that human AGs are composed of pedicle, body, and apex, and are surrounded by a capsule of connective tissues composed of bundles of collagen fibers that form pores of different shapes and sizes. In addition, there are unique connections between arachnoid cells to facilitate the flow of CSF [8]. However, the role of AGs in CSF circulation is controversial. According to some research, most CSF flows out through lymphatic vessels associated with the extracranial segments of cranial nerves.

Acute obstructive hydrocephalus and chronic communicating hydrocephalus may occur after SAH. Obstructive hydrocephalus mainly occurs within three days after SAH and is caused by sudden obstruction of CSF circulation, accompanied by acute intracranial pressure increase and consciousness disorders. Communicating hydrocephalus mainly occurs two weeks after SAH, and it typically manifests itself as mental retardation, gait anomalies, and urinary incontinence. The pathogenesis of hydrocephalus after SAH has not been fully elucidated. Early researchers found that arachnoid cell proliferation caused by inflammation or blood coagulation products can block CSF flow through AGs into venous sinuses. With the emergence of a large number of studies on hydrocephalus after SAH since the 1970s [9, 10], more and more evidence supports that the inflammatory reaction and subsequent fibrotic process impede the outflow of CSF from AGs to sinuses. This research mainly focuses on the pathological obstruction of AGs, including mechanical obstruction and fibrosis of AGs. Previous studies have proposed that blood clots play an initial role in triggering the excessive secretion of CSF and fibrosis of arachnoid granulation, thereby causing long-term communicating hydrocephalus rather than mere aqueduct obstruction or stenosis [11]. With the emergence of abundant evidence of fibrotic pathways, it is generally believed that the fibrosis and adhesion of leptomeningeal and AGs cause chronic communicating hydrocephalus. Red blood cell metabolites, especially iron, have long been assumed to play important roles in the pathophysiological processes of chronic hydrocephalus after SAH. Previous studies suggested that iron oxidation accounts for the precise mechanisms of pathogenesis of hydrocephalus [12], initially termed “ferroptosis” [13]. Intraventricular injection of iron (ferrous chloride or ferric chloride) or lysed red blood cells into rats will lead to hydrocephalus [14]. In addition, iron also causes necrosis of brain cells and disruption of the blood-brain barrier in rats [15], which adds to the strength of this hypothesis. In this research, cynomolgus monkeys were used as research subjects due to their similar anatomy to humans. Repeated injection of autologous blood into the cisterna magna led to hydrocephalus, and other results confirmed extensive fibrosis of AGs. Our results further confirmed the above theory, but more evidence is still needed to further clarify the connection between “ferroptosis” and hydrocephalus.

We look forward to a credible clinical trial to establish whether the removal of blood clots or subarachnoid blood lavage in the initial stage of SAH will produce positive results for these patients.

Regarding research limitations, the observed subjects were insufficient, which may cause biased results, so repeating the experiment with larger sample sizes is necessary. Furthermore, we only monitored the morphological changes of arachnoid particles. To prove the correlation between iron and the pathogenesis of hydrocephalus, quantitative research on iron and other inflammation-related factors in AGs should be conducted.

YL guaranteed the integrity of the entire study. LM designed the study. HY, JZ, and YJ performed the research. YJ analyzed the data. YJ, JY, and YL wrote the manuscript. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript.

This experiment was conducted in Shandong Hongli Medical Animal Experimental Research Co., Ltd., under the supervision of the Animal Ethics Committee of Shandong Hongli Medical Animal Experimental Research Co., Ltd. All operations in this experiment complied with “Regulations on the Administration of Laboratory Animals” of the People’s Republic of China.

We would like to thank all the reviewers for their opinions and suggestion.

This work was supported by Jinan outstanding talent fund sponsored project (2015).

The authors declare no conflict of interest.